AU767147B2 - Ebola virion proteins expressed from Venezuelan equine encephalitis (VEE) virus replicons - Google Patents

Description

WO 00/00617 PCT/US99/14311 1 1 TITLE OF THE INVENTION 2 3 Ebola Virion Proteins Expressed from Venezuelan Equine 4 Encephalitis (VEE) Virus Replicons 6 7 8 9 11

INTRODUCTION

12 13 Ebola viruses, members of the family 14 Filoviridae, are associated with outbreaks of highly lethal hemorrhagic fever in humans and nonhuman 16 primates.. The natural reservoir of the virus is 17 unknown and there currently are no available vaccines 18 or effective therapeutic treatments for filovirus 19 infections. The genome of Ebola virus consists of a single strand of negative sense RNA that is 21 approximately 19 kb in length. This RNA contains seven 22 sequentially arranged genes that produce 8 mRNAs upon 23 infection (Fig. Ebola virions, like virions of 24 other filoviruses, contain seven proteins: a surface glycoprotein a nucleoprotein four virion 26 structural proteins (VP40, VP35, VP30, and VP24), and 27 an RNA-dependent RNA polymerase (Feldmann et 28 al.(1992) Virus Res. 24, 1-19; Sanchez et al.,(1993) 29 Virus Res. 29, 215-240; reviewed in Peters et al.

(1996) In Fields Virology, Third ed. pp. 1161-1176.

31 Fields, B. Knipe, D. Howley, et al. eds.

32 Lippincott-Raven Publishers, Philadelphia). The 33 glycoprotein of Ebola virus is unusual in that it is 34 encoded in two open reading frames. Transcriptional editing is needed to express the transmembrane form 36 that is incorporated into the virion (Sanchez et al.

37 (1996) Proc. Natl. Acad. Sci. USA 93, 3602-3607; WO 00/00617 PCTIUS99/1 4311 2 1 Volchkov et al, (1995) Virology 214, 421-430. The 2 unedited form produces a nonstructural secreted 3 glycoprotein (sGP) that is synthesized in large 4 amounts early during the course of infection. Little is known about the biological functions of these 6 proteins and it is not known which antigens 7 significantly contribute to protection and should 8 therefore be used to induce an immune response.

9 Recent studies using rodent models to evaluate subunit vaccines for Ebola virus infection using 11 recombinant vaccinia virus encoding Ebola virus GP 12 (Gilligan et al., (1997) In Vaccines 97, pp. 87-92.

13 Cold Spring Harbor Laboratory Press, Cold Spring 14 Harbor, or naked DNA constructs expressing either GP or sGP (Xu et al. (1998) Nature Med. 4, 37- 16 42) have demonstrated the protective efficacy of Ebola 17 virus GP in guinea pigs. (All documents cited herein 18 supra and infra are hereby incorporated in their 19 entirety by reference thereto.) Additionally, Ebola virus NP and GP genes expressed from naked DNA 21 vaccines (Vanderzanden et al.,(1998) Virology 246, 22 134-144) have elicited protective immunity in BALB/c 23 mice. However, successful vaccination of nonhuman 24 primates with individual Ebola virus genes has not been demonstrated. Therefore, there exists a need for 26 a vaccine which is efficacious for protection from 27 Ebola virus infection.

28 29 SUMMARY OF THE INVENTION The present invention satisfies the need 31 discussed above. The present invention relates to a 32 method and composition for use in inducing an immune 33 response which is protective against infection with 34 Ebola virus.

Because the biological functions of the 36 individual Ebola virus proteins are not known and the 37 immune mechanisms necessary for preventing and WO 00/00617 PCT/US99/14311 3 1 clearing Ebola virus infection are not well 2 understood, it was not clear which antigens 3 significantly contribute to protection and should 4 therefore be included in an eventual vaccine candidate to induce a protective immune response. We evaluated 6 the ability of packaged Venezuelan equine encephalitis 7 (VEE) virus replicons expressing GP, NP, VP40, 8 VP30 and VP24 virion proteins of Ebola virus to elicit 9 protective immunity in two strains of mice which differ at the major histocompatibility locus. There 11 are no published reports of the VP proteins having 12 been assayed as antigens for the production of an 13 immune response in a mammal.

14 The VEE virus replicon (Vrep) is a genetically reorganized version of the VEE virus genome in which 16 the structural protein genes are replaced with a gene 17 from an immunogen of interest, such as the Ebola virus 18 virion proteins. This replicon can be transcribed to 19 produce a self-replicating RNA that can be packaged into infectious particles using defective helper RNAs 21 that encode the glycoprotein and capsid proteins of 22 the VEE virus. Since the packaged replicons do not 23 encode the structural proteins, they are incapable of 24 spreading to new cells and therefore undergo a single abortive round of replication in which large amounts 26 of the inserted immunogen are made in the infected 27 cells. The VEE virus replicon system is described in 28 U.S. Patent to Johnston et al., patent no. 5,792,462 29 issued on August 11, 1998.

For our purposes, each of the Ebola virus genes 31 were individually inserted into a VEE virus replicon 32 vector. The VP24, VP30, VP35, and VP40 genes of Ebola 33 Zaire 1976 (Mayinga isolate) were cloned by reverse 34 transcription of RNA from Ebola-infected Vero E6 cells and viral cDNAs were amplified using the polymerase 36 chain reaction. The Ebola Zaire 1976 (Mayinga isolate) 37 GP and NP genes were obtained from plasmids already 38 containing these genes (Sanchez, A. et al., (1989) WO 00/00617 PCT/US99/14311 4 1 Virology 170, 81-91; Sanchez, A. et al.,(1993) Virus 2 Res. 29, 215-240) and were subcloned into the VEE 3 replicon vector.

4 After characterization of the Ebola gene products expressed from the VEE replicon constructs in 6 cell culture, these constructs were packaged into 7 infectious VEE virus replicon particles (VRPs) and 8 subcutaneously injected into BALB/c and C57BL/6 mice.

9 As controls in these experiments, mice were also immunized with a VEE replicon expressing Lassa 11 nucleoprotein (NP) as an irrelevant control antigen, 12 or injected with PBS buffer alone. The results of this 13 study demonstrate that VRPs expressing the Ebola GP, 14 NP, VP24, VP30, VP35 or VP40 genes induced protection in mice and may provide protection in humans.

16 17 Therefore, it is one object of the present 18 invention to provide a DNA fragment encoding each of 19 the Ebola Zaire 1976 GP, NP, VP24, VP30, VP35, and VP40 virion proteins(SEQUENCE ID NOS. 1-7).

21 22 It is another object of the present invention to 23 provide the DNA fragments of Ebola virion proteins in 24 a recombinant vector. When the vector is an expression vector, the Ebola virion proteins GP, NP, 26 VP24, VP30, VP35, and VP40 are produced.

27 28 It is yet another object of the present 29 invention to provide a VEE virus replicon vector comprising a VEE virus replicon and a DNA fragment 31 encoding any of the Ebola Zaire 1976 (Mayinga isolate) 32 GP, NP, VP24, VP30, VP35, or VP40 proteins. The 33 construct can be used as a nucleic acid vaccine or for 34 the production of self replicating RNA.

36 It is another object of the present invention to 37 provide a self replicating RNA comprising the VEE 38 virus replicon and any of the Ebola Zaire 1976 WO 00/00617 PCT/US99/14311 1 (Mayinga isolate) RNAs encoding the GP, NP, VP24, 2 VP30, VP35, and VP40 proteins described above. The 3 RNA can be used as a vaccine for protection from Ebola 4 infection. When the RNA is packaged, a VEE virus replicon particle is produced.

6 7 It is another object of the present invention to 8 provide infectious VEE virus replicon particles 9 produced from the VEE virus replicon RNAs described above.

11 12 It is further an object of the invention to 13 provide an immunological composition for the 14 protection of subjects against Ebola virus infection, comprising VEE virus replicon particles containing the 16 Ebola virus GP, NP, VP24, VP30, VP35, or 17 proteins, or any combination of different VEE virus 18 replicons each containing one or more different Ebola 19 proteins selected from GP, NP, VP24, VP30, VP35 and 21 22 BRIEF DESCRIPTION OF THE DRAWINGS 23 These and other features, aspects, and 24 advantages of the present invention will become better understood with reference to the following description 26 and appended claims, and accompanying drawings where: 27 Figure 1 is a schematic description of the 28 organization of the Ebola virus genome.

29 Figures 2A, 2B and 2C are schematic representations of the VEE replicon constructs 31 containing Ebola genes.

32 Figure 3 shows the generation of VEE viral-like 33 particles containing Ebola genes.

34 Figure 4 is an immunoprecipitation of Ebola proteins produced from replicon constructs.

36 WO 00/00617 PCTIUS99/1 4311 6 2 DETAILED DESCRIPTION 3 In the description that follows, a number of 4 terms used in recombinant DNA, virology and immunology are extensively utilized. In order to provide a 6 clearer and consistent understanding of the 7 specification and claims, including the scope to be 8 given such terms, the following definitions are 9 provided.

Filoviruses. The filoviruses Ebola Zaire 11 1976) cause acute hemorrhagic fever characterized by 12 high mortality. Humans can contract filoviruses by 13 infection in endemic regions, by contact with imported 14 primates, and by performing scientific research with the virus. However, there currently are no available 16 vaccines or effective therapeutic treatments for 17 filovirus infection. The virions of filoviruses 18 contain seven proteins: a membrane-anchored 19 glycoprotein a nucleoprotein an RNAdependent RNA polymerase and four virion 21 structural proteins (VP24, VP30, VP35, and 22 Little is known about the biological functions of 23 these proteins and it is not known which antigens 24 significantly contribute to protection and should therefore be used in an eventual vaccine candidate.

26 Replicon. A replicon is equivalent to a full- 27 length virus from which all of the viral structural 28 proteins have been deleted. A multiple cloning site 29 can be inserted downstream of the 26S promoter into the site previously occupied by the structural protein 31 genes. Virtually any heterologous gene may be inserted 32 into this cloning site. The RNA that is transcribed 33 from the replicon is capable of replicating and 34 expressing viral proteins in a manner that is similar to that seen with the full-length infectious virus 36 clone. However, in lieu of the viral structural 37 proteins, the heterologous antigen is expressed from WO 00/00617 PCT/US99/1 4311 7 1 the 26S promoter in the replicon. This system does not 2 yield any progeny virus particles because there are no 3 viral structural proteins available to package the RNA 4 into particles.

Particles which appear structurally identical to 6 virus particles can be produced by supplying 7 structural protein RNAs in trans for packaging of the 8 replicon RNA. This is typically done with two 9 defective helper RNAs which encode the structural proteins. One helper consists of a full length 11 infectious clone from which the nonstructural protein 12 genes and the glycoprotein genes are deleted. This 13 helper retains only the terminal nucleotide sequences, 14 the promoter for subgenomic mRNA transcription and the sequences for the viral nucleocapsid protein. The 16 second helper is identical to the first except that 17 the nucleocapsid gene is deleted and only the 18 glycoprotein genes are retained. The helper RNAs are 19 transcribed in vitro and are co-transfected with replicon RNA. Because the replicon RNA retains the 21 sequences for packaging by the nucleocapsid protein, 22 and because the helpers lack these sequences, only the 23 replicon RNA is packaged by the viral structural 24 proteins. The packaged replicon particles are released from the host cell and can then be purified and 26 inoculated into animals. The packaged replicon 27 particles will have a tropism similar to the parent 28 virus. The packaged replicon particles will infect 29 cells and initiate a single round of replication, resulting in the expression of only the virus 31 nonstructural proteins and the product of the 32 heterologous gene that was cloned in the place of the 33 virus structural proteins. In the absence of RNA 34 encoding the virus structural proteins, no progeny virus particles can be produced from the cells 36 infected by packaged replicon particles.

37 The Venezuelan equine encephalitis (VEE) virus 38 replicon is a genetically reorganized version of the WO 00/00617 PCT/US99/14311 8 1 VEE virus genome in which the genes encoding the VEE 2 structural proteins are replaced with a heterologous 3 gene of interest. In the present invention, the 4 heterologous genes are the GP, NP, or VP virion proteins from the Ebola virus. The result is a self- 6 replicating RNA that can be packaged into infectious 7 particles using defective helper RNAs that encode the 8 glycoprotein and capsid proteins of the VEE virus. The 9 replicon and its use is further described in U.S.

Patent no 5,792,462 issued to Johnston et al. on 11 August 11, 1998.

12 Subject. Includes both human, animal, e.g., 13 horse, donkey, pig, mouse, hamster, monkey, chicken, 14 and insect such as mosquito.

In one embodiment, the present invention relates 16 to DNA fragments which encode any of the Ebola Zaire 17 1976 (Mayinga isolate) GP, NP, VP24, VP30, VP35, and 18 VP40 proteins. The GP and NP genes of Ebola Zaire were 19 previously sequenced by Sanchez et al. (1993, supra) and have been deposited in GenBank (accession number 21 L11365). A plasmid encoding the VEE replicon vector 22 containing a unique Clal site downstream from the 26S 23 promoter was described previously (Davis, N. L. et 24 al., (1996) J. Virol. 70, 3781-3787; Pushko, P. et al. (1997) Virology 239, 389-401). The Ebola GP and 26 NP genes from the Ebola Zaire 1976 virus were derived 27 from PS64- and PGEM3ZF(-)-based plasmids (Sanchez, A.

28 et al. (1989) Virology 170, 81-91; Sanchez, A. et al.

29 (1993) Virus Res. 29, 215-240). From these plasmids, the BamHI-EcoRI (2.3 kb) and BamHI-KpnI (2.4 kb) 31 fragments containing the NP and GP genes, 32 respectively, were subcloned into a shuttle vector 33 that had been digested with BamHI and EcoRI (Davis et 34 al. (1996) supra; Grieder, F. B. et al. (1995) Virology 206, 994-1006). For cloning of the GP gene, 36 overhanging ends produced by KpnI (in the GP fragment) 37 and EcoRI (in the shuttle vector) were made blunt by 38 incubation with T4 DNA polymerase according to methods WO 00/00617 PCT/US99/14311 9 1 known in the art. From the shuttle vector, GP or NP 2 genes were subcloned as Clal-fragments into the Clal 3 site of the replicon clone, resulting in plasmids 4 encoding the GP or NP genes in place of the VEE structural protein genes downstream from the VEE 26S 6 promoter.

7 The VP genes of Ebola Zaire were previously 8 sequenced by Sanchez et al. (1993, supra) and have 9 been deposited in GenBank (accession number L11365).

The VP genes of Ebola used in the present invention 11 were cloned by reverse transcription of RNA from 12 Ebola-infected Vero E6 cells and subsequent 13 amplification of viral cDNAs using the polymerase 14 chain reaction. First strand synthesis was primed with oligo dT (Life Technologies). Second strand synthesis 16 and subsequent amplification of viral cDNAs were 17 performed with gene-specific primers (SEQ ID NOS:8- 18 16). The primer sequences were derived from the 19 GenBank deposited sequences and were designed to contain a Clal restriction site for cloning the 21 amplified VP genes into the Clal site of the replicon 22 vector. The letters and numbers in bold print indicate 23 Ebola gene sequences in the primers and the 24 corresponding location numbers based on the GenBank depositied sequences.

26 VP24: forward primer is 27 5'-GGGATCGATCTCCAGACACCAAGCAAGACC- 3'(SEQ ID NO:8) 28 (10,311-10,331) 29 reverse primer is 5'-GGGATCGATGAGTCAGCATATATGAGTTAGCTC-3' (SEQ ID 31 NO:9) 32 (11,122-11,145) 33 VP30: forward primer is 34 5'-CCCATCGATCAGATCTGCGAACCGGTAGAG-3' SEQ ID (8408-8430) 36 reverse primer is 37 5'-CCCATCGATGTACCCTCATCAGACCATGAGC-3' (SEQ ID 38 NO:11) WO 00/00617 PCT/US99/14311 1 (9347-9368) 2 VP35: forward primer is 3 5'-GGGATCGATAGAAAAGCTGGTCTAACAAGATGA-3'(SEQ

ID

4 NO:12) (3110-3133) 6 reverse primer is 7 5' -CCCATCGATCTCACAAGTGTATCATTAATGTAACGT-3 (SEQ ID 8 NO:13) (4218-4244) 9 VP40: forward primer is 5'-CCCATCGATCCTACCTCGGCTGAGAGAGTG-3'(SEQ ID NO:14) 11 (4408-4428) 12 reverse primer is 13 5'-CCCATCGATATGTTATGCACTATCCCTGAGAAG-3'(SEQ

ID

14 (5495-5518) 16 VP30 #2: 17 forward primer as for VP30 above 18 reverse primer is 19 5'-CCCATCGATCTGTTAGGGTTGTATCATACC-3'(SEQ ID NO:16) 21 The Ebola virus genes cloned into the VEE 22 replicon were sequenced. Changes in the DNA sequence 23 relative to the sequence published by Sanchez et al.

24 (1993) are described relative to the nucleotide (nt) sequence number from GenBank (accession number 26 L11365).

27 The nucleotide sequence we obtained for Ebola 28 virus GP (SEQ ID NO:1) differed from the GenBank 29 sequence by a transition from A to G at nt 8023. This resulted in a change in the amino acid sequence from 31 Ile to Val at position 662 (SEQ ID NO: 17).

32 The nucleotide sequence we obtained for Ebola 33 virus NP (SEQ ID NO:2) differed from the GenBank 34 sequence at the following 4 positions: insertion of a C residue between nt 973 and 974, deletion of a G 36 residue at nt 979, transition from C to T at nt 1307, 37 and a transversion from A to C at nt 2745. These 38 changes resulted in a change in the protein sequence WO 00/00617 PCT/US99/14311 11 1 from Arg to Glu at position 170 and a change from Leu 2 to Phe at position 280 (SEQ ID NO: 18).

3 The Ebola virus VP24 nucleotide sequence (SEQ ID 4 NO:3) differed from the GenBank sequence at 6 positions, resulting in 3 nonconservative changes in 6 the amino acid sequence. The changes in the DNA 7 sequence of VP24 consisted of a transversion from G to 8 C at nt 10795, a transversion from C to G at nt 10796, 9 a transversion from T to A at nt 10846, a transversion from A to T at nt 10847, a transversion from C to G at 11 nt 11040, and a transversion from C to G at nt 11041.

12 The changes in the amino acid sequence of VP24 13 consisted of a Cys to Ser change at position 151, a 14 Leu to His change at position 168, and a Pro to Gly change at position 233 (SEQ ID NO: 19).

16 Two different sequences for the Ebola virus 17 gene, VP30 and VP30#2 (SEQ ID NOS: 4 and 7) are 18 included. Both of these sequences differ from the 19 GenBank sequence by the insertion of an A residue in the upstream noncoding sequence between nt 8469 and 21 8470 and an insertion of a T residue between nt 9275 22 and 9276 that results in a change in the open reading 23 frame of VP30 and VP30#2 after position 255 (SEQ ID 24 NOS: 20 and 23). As a result, the C-terminus of the VP30 protein differs significantly from that 26 previously reported. In addition to these 2 changes, 27 the VP30#2 nucleic acid in SEQ ID NO:7 contains a 28 conservative transition from T to C at nt 9217.

29 Because the primers originally used to clone the gene into the replicon were designed based on the 31 GenBank sequence, the first clone that we constructed 32 (SEQ ID NO: 4) did not contain what we believe to be 33 the authentic C-terminus of the protein. Therefore, 34 in the absence of the VP30 stop codon, the C-terminal codon was replaced with 37 amino acids derived from 36 the vector sequence. The resulting VP30 construct 37 therefore differed from the GenBank sequence in that 38 it contained 32 amino acids of VP30 sequence WO 00/00617 PCT/US99/14311 12 1 (positions 256 to 287, SEQ ID NO:20) and 37 amino 2 acids of irrelevant sequence (positions 288 to 324, 3 SEQ ID NO:20) in the place of the C-terminal 5 amino 4 acids reported in GenBank. However, inclusion of 37 amino acids of vector sequence in place of the C- 6 terminal amino acid (Pro, SEQ ID NO: 23) did not 7 inhibit the ability of the protein to serve as a 8 protective antigen in BALB/c mice. We are currently 9 examining the ability of the new VEE replicon construct, which we believe contains the authentic C- 11 terminus of VP30 (VP30#2, SEQ ID NO: 23), to protect 12 mice against a lethal Ebola challenge.

13 The nucleotide sequence for Ebola virus VP35 (SEQ 14 ID NO:5) differed from the GenBank sequence by a transition from T to C at nt 4006, a transition from T 16 to C at nt 4025, and an insertion of a T residue 17 between nt 4102 and 4103. These sequence changes 18 resulted in a change from a Ser to a Pro at position 19 293 and a change from Phe to Ser at position 299 (SEQ ID NO: 21). The insertion of the T residue resulted 21 in a change in the open reading frame of VP35 from 22 that previously reported by Sanchez et al. (1993) 23 following amino acid number 324. As a result, Ebola 24 virus VP35 encodes a protein of 340 amino acids, where amino acids 325 to 340 (SEQ ID NO: 21) differ from and 26 replace the C-terminal 27 amino acids of the 27 previously published sequence.

28 Sequencing of VP30 and VP35 was also performed 29 on RT/PCR products from RNA derived from cells that were infected with Ebola virus 1976, Ebola virus 1995 31 or the mouse-adapted Ebola virus. The changes noted 32 above for the Vrep constructs were also found in these 33 Ebola viruses. Thus, we believe that these changes are 34 real events and not artifacts of cloning.

The Ebola virus VP40 nucleotide sequence (SEQ ID 36 NO:6) differed from the GenBank sequence by a 37 transversion from a C to G at nt 4451 and a transition 38 from a G to A at nt 5081. These sequence changes did WO 00/00617 PCTIUS99/1 4311 13 I not alter the protein sequence of VP40 (SEQ ID NO: 22) 2 from that of the published sequence.

3 DNA or polynucleotide sequences to which the 4 invention also relates include sequences of at least about 6 nucleotides, preferably at least about 8 6 nucleotides, more preferably at least about 10-12 7 nucleotides, most preferably at least about 15-20 8 nucleotides corresponding, homologous to or 9 complementary to, a region of the Ebola nucleotide sequences described above. Preferably, the sequence of 11 the region from which the polynucleotide is derived is 12 homologous to or complementary to a sequence which is 13 unique to the Ebola genes. Whether or not a sequence is 14 unique to the Ebola gene can be determined by techniques known to those of skill in the art. For example, the 16 sequence can be compared to sequences in databanks, 17 GenBank and compared by DNA:DNA hybridization.

18 Regions from which typical DNA sequences may be derived 19 include but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed 21 and/or non-translated regions.

22 The derived polynucleotide is not necessarily 23 physically derived from the nucleotide sequences shown 24 in SEQ ID NO:I-7, but may be generated in any manner, including for example, chemical synthesis or DNA 26 replication or reverse transcription or transcription, 27 which are based on the information provided by the 28 sequence of bases in the region(s) from which the 29 polynucleotide is derived. In addition, combinations of regions corresponding to that of the designated 31 sequence may be modified in ways known in the art to 32 be consistent with an intended use. The sequences of 33 the present invention can be used in diagnostic assays 34 such as hybridization assays and polymerase chain reaction assays, for example, for the discovery of 36 other Ebola sequences.

37 In another embodiment, the present invention 38 relates to a recombinant DNA molecule that includes a WO 00/00617 PCT/US99/14311 14 1 vector and a DNA sequence as described above. The 2 vector can take the form of a plasmid, a eukaryotic 3 expression vector such as pcDNA3.1, pRcCMV2, 4 pZeoSV2,or pCDM8, which are available from Invitrogen, or a virus vector such as baculovirus vectors, 6 retrovirus vectors or adenovirus vectors, alphavirus 7 vectors, and others known in the art.

8 In a further embodiment, the present invention 9 relates to host cells stably transformed or transfected with the above-described recombinant DNA 11 constructs. The host cell can be prokaryotic (for 12 example, bacterial), lower eukaryotic (for example, 13 yeast or insect) or higher eukaryotic (for example, 14 all mammals, including but not limited to mouse and human). Both prokaryotic and eukaryotic host cells may 16 be used for expression of the desired coding sequences 17 when appropriate control sequences which are 18 compatible with the designated host are used.

19 Among prokaryotic hosts, E. coli is the most frequently used host cell for expression. General 21 control sequences for prokaryotes include promoters 22 and ribosome binding sites. Transfer vectors 23 compatible with prokaryotic hosts are commonly derived 24 from a plasmid containing genes conferring ampicillin and tetracycline resistance (for example, pBR322) or 26 from the various pUC vectors, which also contain 27 sequences conferring antibiotic resistance. These 28 antibiotic resistance genes may be used to obtain 29 successful transformants by selection on medium containing the appropriate antibiotics. Please see 31 Maniatis, Fitsch and Sambrook, Molecular 32 Cloning; A Laboratory Manual (1982) or DNA Cloning, 33 Volumes I and II N. Glover ed. 1985) for general 34 cloning methods. The DNA sequence can be present in the vector operably linked to sequences encoding an 36 IgG molecule, an adjuvant, a carrier, or an agent for WO 00/00617 PCT/US99/14311 1 aid in purification of Ebola proteins, such as 2 glutathione S-transferase.

3 In addition, the Ebola virus gene products can 4 also be expressed in eukaryotic host cells such as yeast cells and mammalian cells. Saccharomyces 6 cerevisiae, Saccharomyces carlsbergensis, and Pichia 7 pastoris are the most commonly used yeast hosts.

8 Control sequences for yeast vectors are known in the 9 art. Mammalian cell lines available as hosts for expression of cloned genes are known in the art and 11 include many immortalized cell lines available from 12 the American Type Culture Collection (ATCC), such as 13 CHO cells, Vero cells, baby hamster kidney (BHK) cells 14 and COS cells, to name a few. Suitable promoters are also known in the art and include viral promoters such 16 as that from SV40, Rous sarcoma virus (RSV), 17 adenovirus (ADV), bovine papilloma virus (BPV), and 18 cytomegalovirus (CMV). Mammalian cells may also 19 require terminator sequences, poly A addition sequences, enhancer sequences which increase 21 expression, or sequences which cause amplification of 22 the gene. These sequences are known in the art.

23 The transformed or transfected host cells can be 24 used as a source of DNA sequences described above.

When the recombinant molecule takes the form of an 26 expression system, the transformed or transfected 27 cells can be used as a source of the protein described 28 below.

29 In another embodiment, the present invention relates to Ebola virion proteins such as GP having an 31 amino acid sequence corresponding to SEQ ID NO:17 32 encompassing 676 amino acids, NP, having an amino acid 33 sequence corresponding to SEQ ID NO:18 encompassing 34 739 amino acids, VP24, having an amino acid sequence corresponding to SEQ ID NO:19 encompassing 251 amino 36 acids, VP30, having an amino acid sequence 37 corresponding SEQ ID NO:20 encompassing 324 amino 38 acids, VP35, having an amino acid sequence WO 00/00617 PCT/US99/14311 16 1 corresponding to SEQ ID NO:21 encompassing 340 amino 2 acids, and VP40, having an amino acid sequence 3 corresponding to SEQ ID NO:22, encompassing 326 amino 4 acids, and VP30#2, having an amino acid sequence corresponding to SEQ ID NO:23 encompassing 288 amino 6 acids, or any allelic variation of the amino acid 7 sequences. By allelic variation is meant a natural or 8 synthetic change in one or more amino acids which 9 occurs between different serotypes or strains of Ebola virus and does not affect the antigenic properties of 1I the protein. There are different strains of Ebola 12 (Zaire 1976, Zaire 1995, Reston, Sudan, and Ivory 13 Coast). The NP and VP genes of these different viruses 14 have not been sequenced. It would be expected that these proteins would have homology among different 16 strains and that vaccination against one Ebola virus 17 strain might afford cross protection to other Ebola 18 virus strains.

19 A polypeptide or amino acid sequence derived from any of the amino acid sequences in SEQ ID NO:17, 21 18, 19, 20, 21, 22, and 23 refers to a polypeptide 22 having an amino acid sequence identical to that of a 23 polypeptide encoded in the sequence, or a portion 24 thereof wherein the portion consists of at least amino acids, preferably at least 8-10 amino acids, and 26 more preferably at least 11-15 amino acids, or which 27 is immunologically identifiable with a polypeptide 28 encoded in the sequence.

29 A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid 31 sequence, or the DNA sequence found in GenBank 32 accession number L11365. It may be generated in any 33 manner, including for example, chemical synthesis, or 34 expression from a recombinant expression system.

When the DNA or RNA sequences described above 36 are in a replicon expression system, such as the VEE 37 replicon described above, the proteins can be 38 expressed in vivo. The DNA sequence for any of the WO 00/00617 PCTIUS99/14311 17 1 GP, NP, VP24, VP30, VP35, and VP40 virion proteins can 2 be cloned into the multiple cloning site of a replicon 3 such that transcription of the RNA from the replicon 4 yields an infectious RNA encoding the Ebola protein or proteins of interest (see Figure 2A, 2B and 2C). The 6 replicon constructs include Ebola virus GP (SEQ ID 7 NO:1) cloned into a VEE replicon (VRepEboGP), Ebola 8 virus NP (SEQ ID NO:2) cloned into a VEE replicon 9 (VRepEboNP), Ebola virus VP24 (SEQ ID NO:3) cloned into a VEE replicon (VRepEboVP24), Ebola virus 11 (SEQ ID NO:4) or VP30#2 (SEQ ID NO:7) cloned into a 12 VEE replicon (VRepEboVP30 or VRepEboVP30(#2)), Ebola 13 virus VP35 (SEQ ID NO:5) cloned into a VEE replicon 14 (VRepEboVP35), and Ebola virus VP40 (SEQ ID NO:6) cloned into a VEE replicon (VRepEboVP40). The 16 replicon DNA or RNA can be used as a vaccine for 17 inducing protection against infection with Ebola.

18 Use of helper RNAs containing sequences necessary for 19 packaging of the viral replicon transcripts will result in the production of virus-like particles 21 containing replicon RNAs (Figure These packaged 22 replicons will infect host cells and initiate a single 23 round of replication resulting in the expression of 24 the Ebola proteins in said infected cells. The packaged replicon constructs VEE virus replicon 26 particles, VRP) include those that express Ebola virus 27 GP (EboGPVRP), Ebola virus NP (EboNPVRP), Ebola virus 28 VP24 (EboVP24VRP), Ebola virus VP30 (EboVP30VRP or 29 EboVP30VRP(#2)), Ebola virus VP35 (EboVP35VRP), and Ebola virus VP40 31 In another embodiment, the present invention 32 relates to RNA molecules resulting from the 33 transcription of the constructs described above. The 34 RNA molecules can be prepared by in vitro transcription using methods known in the art and described in the 36 Examples below. Alternatively, the RNA molecules can be 37 produced by transcription of the constructs in vivo, and 38 isolating the RNA. These and other methods for WO 00/00617 PCT/US99/14311 18 1 obtaining RNA transcripts of the constructs are known in 2 the art. Please see Current Protocols in Molecular 3 Biolocv. Frederick M. Ausubel et al. John Wiley 4 and Sons, Inc. The RNA molecules can be used, for example, as a direct RNA vaccine, or to transfect cells 6 along with RNA from helper plasmids, one of which 7 expresses VEE glycoproteins and the other VEE capsid 8 proteins, as described above, in order to obtain 9 replicon particles.

In a further embodiment, the present invention 11 relates to a method of producing the recombinant or 12 fusion protein which includes culturing the above- 13 described host cells under conditions such that the 14 DNA fragment is expressed and the recombinant or fusion protein is produced thereby. The recombinant or 16 fusion protein can then be isolated using methodology 17 well known in the art. The recombinant or fusion 18 protein can be used as a vaccine for immunity against 19 infection with Ebola or as a diagnostic tool for detection of Ebola infection.

21 In another embodiment, the present invention 22 relates to antibodies specific for the above-described 23 recombinant proteins (or polypeptides). For instance, 24 an antibody can be raised against a peptide having the amino acid sequence of any of SEQ ID N0:17-25, or 26 against a portion thereof of at least 10 amino acids, 27 preferably, 11-15 amino acids. Persons with ordinary 28 skill in the art using standard methodology can raise 29 monoclonal and polyclonal antibodies to the protein(or polypeptide) of the present invention, or a unique 31 portion thereof. Materials and methods for producing 32 antibodies are well known in the art (see for example 33 Goding, In Monoclonal Antibodies: Principles and 34 Practice, Chapter 4, 1986).

In a further embodiment, the present invention 36 relates to a method of detecting the presence of 37 antibodies against Ebola virus in a sample. Using WO 00/00617 PCT[US99/1 4311 19 1 standard methodology well known in the art, a 2 diagnostic assay can be constructed by coating on a 3 surface a solid support for example, a 4 microtitration plate, a membrane nitrocellulose membrane) or a dipstick), all or a unique portion of 6 any of the Ebola proteins described above or any 7 combination thereof, and contacting it with the serum 8 of a person or animal suspected of having Ebola. The 9 presence of a resulting complex formed between the Ebola protein(s) and serum antibodies specific 11 therefor can be detected by any of the known methods 12 common in the art, such as fluorescent antibody 13 spectroscopy or colorimetry. This method of detection 14 can be used, for example, for the diagnosis of Ebola infection and for determining the degree to which an 16 individual has developed virus-specific Abs after 17 administration of a vaccine.

18 In yet another embodiment, the present invention 19 relates to a method for detecting the presence of Ebola virion proteins in a sample. Antibodies against 21 GP, NP, and the VP proteins could be used for 22 diagnostic assays. Using standard methodology well 23 known in the art, a diagnostics assay can be 24 constructed by coating on a surface a solid support, for example, a microtitration plate or a 26 membrane nitrocellulose membrane)), antibodies 27 specific for any of the Ebola proteins described 28 above, and contacting it with serum or a tissue sample 29 of a person suspected of having Ebola infection. The presence of a resulting complex formed between the 31 protein or proteins in the serum and antibodies 32 specific therefor can be detected by any of the known 33 methods common in the art, such as fluorescent 34 antibody spectroscopy or colorimetry. This method of detection can be used, for example, for the diagnosis 36 of Ebola virus infection.

37 In another embodiment, the present invention 38 relates to a diagnostic kit which contains any WO 00/00617 PCT/US99/14311 1 combination of the Ebola proteins described above and 2 ancillary reagents that are well known in the art and 3 that are suitable for use in detecting the presence of 4 antibodies to Ebola in serum or a tissue sample.

Tissue samples contemplated can be from monkeys, 6 humans, or other mammals.

7 In yet another embodiment, the present invention 8 relates to DNA or nucleotide sequences for use in 9 detecting the presence of Ebola virus using the reverse transcription-polymerase chain reaction (RT- 11 PCR). The DNA sequence of the present invention can 12 be used to design primers which specifically bind to 13 the viral RNA for the purpose of detecting the 14 presence of Ebola virus or for measuring the amount of Ebola virus in a sample. The primers can be any 16 length ranging from 7 to 400 nucleotides, preferably 17 at least 10 to 15 nucleotides, or more preferably 18 18 to 40 nucleotides. Reagents and controls necessary 19 for PCR reactions are well known in the art. The amplified products can then be analyzed for the 21 presence of viral sequences, for example by gel 22 fractionation, with or without hybridization, by 23 radiochemistry, and immunochemistry techniques.

24 In yet another embodiment, the present invention relates to a diagnostic kit which contains PCR primers 26 specific for Ebola virus and ancillary reagents for 27 use in detecting the presence or absence of Ebola in a 28 sample using PCR. Samples contemplated can be obtained 29 from human, animal, horse, donkey, pig, mouse, hamster, monkey, or other mammals, birds, and insects, 31 such as mosquitoes.

32 In another embodiment, the present invention 33 relates to an Ebola vaccine comprising VRPs that 34 express one or more of the Ebola proteins described above. The vaccine is administered to a subject 36 wherein the replicon is able to initiate one round of 37 replication producing the Ebola proteins to which a WO 00/00617 PCT/US99/14311 21 1 protective immune response is initiated in said 2 subject.

3 It is likely that the protection afforded by 4 these genes is due to both the humoral (antibodies (Abs)) and cellular (cytotoxic T cells (CTLs)) arms of 6 the immune system. Protective immunity induced to a 7 specific protein may comprise humoral immunity, 8 cellular immunity, or both. The only Ebola virus 9 protein known to be on the outside of the virion is the GP. The presence of GP on the virion surface 11 makes it a likely target for GP-specific Abs that may 12 bind either extracellular virions or infected cells 13 expressing GP on their surfaces. Serum transfer 14 studies in this invention demonstrate that Abs that recognize GP protect mice against lethal Ebola virus 16 challenge.

17 In contrast, transfer of Abs specific for NP, 18 VP24, VP30, VP35, or VP40 did not protect mice against 19 lethal Ebola challenge. This data, together with the fact that these are internal virion proteins that are 21 not readily accessible to Abs on either extracellular 22 virions or the surface of infected cells, suggest that 23 the protection induced in mice by these proteins is 24 mediated by CTLs.

CTLs can bind to and lyse virally infected cells.

26 This process begins when the proteins produced by 27 cells are routinely digested into peptides. Some of 28 these peptides are bound by the class I or class II 29 molecules of the major histocompatability complex (MHC), which are then transported to the cell surface.

31 During virus infections, viral proteins produced 32 within infected cells also undergo this process. CTLs 33 that have receptors that bind to both a specific 34 peptide and the MHC molecule holding the peptide lyse the peptide-bearing cell, thereby limiting virus 36 replication. Thus, CTLs are characterized as being 37 specific for a particular peptide and restricted to a 38 class I or class II MHC molecule.

WO 00/00617 PCT/US99/14311 22 1 CTLs may be induced against any of the Ebola 2 virus proteins, as all of the viral proteins are 3 produced and digested within the infected cell. Thus, 4 protection to Ebola virus could involve CTLs against GP, NP, VP24, VP30, VP35, and/or VP40. It is 6 especially noteworthy that the VP proteins varied in 7 their protective efficacy when tested in genetically 8 inbred mice that differ at the MHC locus. This, 9 together with the inability to demonstrate a role for Abs in protection induced by the VP proteins, strongly 11 supports a role for CTLs. These data also suggest 12 that an eventual vaccine candidate may include several 13 Ebola virus proteins, or several CTL epitopes, capable 14 of inducing broad protection in outbred populations people). We have identified two sequences 16 recognized by CTLs. They are Ebola virus NP SEQ ID 17 NO:24 and Ebola virus VP24 SEQ ID NO:25. Testing is 18 in progress to identify the role of CTLs in protection 19 induced by each of these Ebola virus proteins and to define the minimal sequence requirements for the 21 protective response. The CTL assay is well known in 22 the art.

23 An eventual vaccine candidate might 24 comprise these CTL sequences and others. These might be delivered as synthetic peptides, or as fusion 26 proteins, alone or co-administered with cytokines 27 and/or adjuvants or carriers safe for human use, e.g.

28 aluminum hydroxide, to increase immunogenicity. In 29 addition, sequences such as ubiquitin can be added to increase antigen processing for more effective CTL 31 responses.

32 In yet another embodiment, the present invention 33 relates to a method for providing immunity against 34 Ebola virus, said method comprising administering one or more VRPs expressing any combination of the GP, NP, 36 VP24, VP30 or VP30#2, VP35 and VP40 Ebola proteins to 37 a subject such that a protective immune reaction is 38 generated.

WO 00/00617 PCT/US99/1 4311 23 I Vaccine formulations of the present invention 2 comprise an immunogenic amount of a VRP, such as for 3 example EboVP24VRP described above, or, for a 4 multivalent vaccine, a combination of replicons, in a pharmaceutically acceptable carrier. An "immunogenic 6 amount" is an amount of the VRP(s) sufficient to evoke 7 an immune response in the subject to which the vaccine 8 is administered. An amount of from about 104-108 9 focus-forming units per dose is suitable, depending upon the age and species of the subject being treated.

11 The subject may be inoculated 2-3 times. Exemplary 12 pharmaceutically acceptable carriers include, but are 13 not limited to, sterile pyrogen-free water and sterile 14 pyrogen-free physiological saline solution.

Administration of the VRPs disclosed herein may 16 be carried out by any suitable means, including 17 parenteral injection (such as intraperitoneal, 18 subcutaneous, or intramuscular injection), in ovo 19 injection of birds, orally, or by topical application of the virus (typically carried in a pharmaceutical 21 formulation) to an airway surface. Topical application 22 of the virus to an airway surface can be carried out 23 by intranasal administration by use of dropper, 24 swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the 26 virus to an airway surface can also be carried out by 27 inhalation administration, such as by creating 28 respirable particles of a pharmaceutical formulation 29 (including both solid particles and liquid particles) containing the replicon as an aerosol suspension, and 31 then causing the subject to inhale the respirable 32 particles. Methods and apparatus for administering 33 respirable particles of pharmaceutical formulations 34 are well known, and any conventional technique can be employed. Oral administration may be in the form of 36 an ingestable liquid or solid formulation.

WO 00/00617 PCT[US99/1 4311 24 I When the replicon RNA or DNA is used as a vaccine, 2 the replicon RNA or DNA can be administered directly 3 using techniques such as delivery on gold beads (gene 4 gun), delivery by liposomes, or direct injection, among other methods known to people in the art. Any one or 6 more DNA constructs or replicating RNA described above 7 can be use in any combination effective to elicit an 8 immunogenic response in a subject. Generally, the 9 nucleic acid vaccine administered may be in an amount of about 1-5 ug of nucleic acid per dose and will depend on II the subject to be treated, capacity of the subject's 12 immune system to develop the desired immune response, 13 and the degree of protection desired. Precise amounts 14 of the vaccine to be administered may depend on the judgement of the practitioner and may be peculiar to 16 each subject and antigen.

17 The vaccine may be given in a single dose 18 schedule, or preferably a multiple dose schedule in 19 which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at 21 subsequent time intervals required to maintain and or 22 reinforce the immune response, for example, at 1-4 23 months for a second dose, and if needed, a subsequent 24 dose(s) after several months. Examples of suitable immunization schedules include: 0, 1 months and 6 26 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 27 month, (iv) 0 and 6 months, or other schedules 28 sufficient to elicit the desired immune responses 29 expected to confer protective immunity, or reduce disease symptoms, or reduce severity of disease.

31 The following examples are included to demonstrate 32 preferred embodiments of the invention. It should be 33 appreciated by those of skill in the art that the 34 techniques disclosed in the examples which follow represent techniques discovered by the inventors and 36 thought to function well in the practice of the 37 invention, and thus can be considered to constitute 38 preferred modes for its practice. However, those of WO 00/00617 PCT/US99/14311 1 skill in the art should, in light of the present 2 disclosure, appreciate that many changes can be made in 3 the specific embodiments which are disclosed and still 4 obtain a like or similar result without departing from the spirit and scope of the invention.

6 7 The following MATERIALS AND METHODS were used in 8 the examples that follow.

9 Cells lines and viruses BHK (ATCC CCL 10), Vero 76 (ATCC CRL 1587), and 11 Vero E6 (ATCC CRL 1586) cell lines were maintained in 12 minimal essential medium with Earle's salts, 5-10% 13 fetal bovine serum, and 50 g/mL gentamicin sulfate.

14 For CTL assays, EL4 (ATCC TIB39), L5178Y (ATCC CRL 1723) and P815 (ATCC TIB64) were maintained in 16 Dulbecco's minimal essential medium supplemented with 17 5-10% fetal bovine serum and antibiotics.

18 A stock of the Zaire strain of Ebola virus 19 originally isolated from a patient in the 1976 outbreak (Mayinga) and passaged intracerebrally 3 21 times in suckling mice and 2 times in Vero cells was 22 adapted to adult mice through serial passage in 23 progressively older suckling mice (Bray et al.,(1998) 24 J. Infect. Dis. 178, 651-661). A plaque-purified ninth-mouse-passage isolate which was uniformly lethal 26 for adult mice ("mouse-adapted virus") was propagated 27 in Vero E6 cells, aliquotted, and used in all mouse 28 challenge experiments and neutralization assays.

29 A stock of the Zaire strain of Ebola 1976 virus was passaged spleen to spleen in strain 13 guinea pigs 31 four times. This guinea pig-adapted strain was used 32 to challenge guinea pigs.

33 Construction and packaging of recombinant VEE 34 virus replicons (VRPs) Replicon RNAs were packaged into VRPs as 36 described (Pushko et al., 1997, supra). Briefly, 37 capped replicon RNAs were produced in vitro by T7 run- WO 00/00617 PCTIUS99/14311 26 1 off transcription of NotI-digested plasmid templates 2 using the RiboMAX T7 RNA polymerase kit (Promega).

3 BHK cells were co-transfected with the replicon RNAs 4 and the 2 helper RNAs expressing the structural proteins of the VEE virus. The cell culture 6 supernatants were harvested approximately 30 hours 7 after transfection and the replicon particles were 8 concentrated and purified by centrifugation through a 9 20% sucrose cushion. The pellets containing the packaged replicon particles were suspended in PBS and 11 the titers were determined by infecting Vero cells 12 with serial dilutions of the replicon particles and 13 enumerating the infected cells by indirect 14 immunofluorescence with antibodies specific for the Ebola proteins.

16 Immunoprecipitation of Ebola virus proteins 17 expressed from VEE virus replicons 18 BHK cells were transfected with either the Ebola 19 virus GP, NP, VP24, VP30, VP35, or VP40 replicon RNAs.

At 24 h post-transfection, the culture medium was 21 replaced with minimal medium lacking cysteine and 22 methionine, and proteins were labeled for 1 h with 23 3 S-labeled methionine and cysteine. Cell lysates or 24 supernatants (supe) were collected and immunoprecipitated with polyclonal rabbit anti-Ebola 26 virus serum bound to protein A beads. 3 S-labeled 27 Ebola virus structural proteins from virions grown in 28 Vero E6 cells were also immunoprecipitated as a 29 control for each of the virion proteins.

Immunoprecipitated proteins were resolved by 31 electrophoresis on an 11% SDS-polyacrylamide gel and 32 were visualized by autoradiography.

33 Vaccination of Mice With VEE Virus Replicons 34 Groups of 10 BALB/c or C57BL/6 mice per experiment were subcutaneously injected at the base of the neck 36 with 2 x 106 focus-forming units of VRPs encoding the 37 Ebola virus genes. As controls, mice were also WO 00/00617 PCT/US99/14311 27 1 injected with either a control VRP encoding the Lassa 2 nucleoprotein (NP) or with PBS. For booster 3 inoculations, animals received identical injections at 4 1 month intervals. Data are recorded as the combined results of 2 or 3 separate experiments.

6 Ebola Infection of Mice 7 One month after the final booster inoculation, 8 mice were transferred to a BSL-4 containment area and 9 challenged by intraperitoneal (ip) inoculation of plaque-forming units (pfu) of mouse-adapted Ebola 11 virus (approximately 300 times the dose lethal for 12 of adult mice). The mice were observed daily, and 13 morbidity and mortality were recorded. Animals 14 surviving at day 21 post-infection were injected again with the same dose of virus and observed for another 16 21 days.

17 In some experiments, 4 or 5 mice from vaccinated 18 and control groups were anesthetized and exsanguinated 19 on day 4 (BALB/c mice) or day 5 (C57BL/6 mice) following the initial viral challenge. The viral 21 titers in individual sera were determined by plaque 22 assay.

23 Passive Transfer Of Immune Sera to Naive Mice.

24 Donor sera were obtained 28 days after the third inoculation with 2 x 106 focus-forming units of VRPs 26 encoding the indicated Ebola virus gene, the control 27 Lassa NP gene, or from unvaccinated control mice. One 28 mL of pooled donor sera was administered 29 intraperitoneally (ip) to naive, syngeneic mice 24 h prior to intraperitoneal challenge with 10 pfu of 31 mouse-adapted Ebola virus.

32 Vaccination and Challenge of Guinea Pigs.

33 EboGPVRP or EboNPVRP (1x10 7 focus-forming units 34 in 0.5ml PBS) were administered subcutaneously to inbred strain 2 or strain 13 guinea pigs (300-400g).

36 Groups of five guinea pigs were inoculated on days 0 37 and 28 at one (strain 2) or two (strain 13) dorsal WO 00/00617 PCTIUS99/14311 28 1 sites. Strain 13 guinea pigs were also boosted on day 2 126. One group of Strain 13 guinea pigs was 3 vaccinated with both the GP and NP constructs. Blood 4 samples were obtained after vaccination and after viral challenge. Guinea pigs were challenged on day 6 56 (strain 2) or day 160 (strain 13) by subcutaneous 7 administration of 1000 LD 50 (1 x 104 PFU) of guinea 8 pig-adapted Ebola virus. Animals were observed daily 9 for 60 days, and morbidity (determined as changes in behavior, appearance, and weight) and survival were 11 recorded. Blood samples were taken on the days 12 indicated after challenge and viremia levels were 13 determined by plaque assay.

14 Virus titration and neutralization assay. Viral stocks were serially diluted in growth medium, 16 adsorbed onto confluent Vero E6 cells in 6- or 12-well 17 dishes, incubated for 1 hour at 37 0 C, and covered with 18 an agarose overlay (Moe, J. et al. (1981) J. Clin.

19 Microbiol. 13:791-793). A second overlay containing neutral red solution in PBS or agarose was added 6 21 days later, and plaques were counted the following 22 day. Pooled pre-challenge serum samples from some of 23 the immunized groups were tested for the presence of 24 Ebola-neutralizing antibodies by plaque reduction neutralization assay. Aliquots of Ebola virus in 26 growth medium were mixed with serial dilutions of test 27 serum, or with normal serum, or medium only, incubated 28 at 37 0 C for 1 h, and used to infect Vero E6 cells.

29 Plaques were counted 1 week later.

Cvtotoxic T cell assays. BALB/c and C57BL/6 mice 31 were inoculated with VRPs encoding Ebola virus NP or 32 VP24 or the control Lassa NP protein. Mice were 33 euthanized at various times after the last inoculation 34 and their spleens removed. The spleens were gently ruptured to generate single cell suspensions. Spleen 36 cells (1 x 106/ ml) were cultured in vitro for 2 days 37 in the presence of 10-25 pM of peptides synthesized WO 00/00617 PCT/US99/14311 29 1 from Ebola virus NP or VP24 amino acid sequences, and 2 then for an additional 5 days in the presence of 3 peptide and 10% supernatant from concanavalin A- 4 stimulated syngeneic spleen cells. Synthetic peptides were made from Ebola virus amino acid sequences 6 predicted by a computer algorithm (HLA Peptide Binding 7 Predictions, Parker, K. et al. (1994) J. Immunol.

8 152:163) to have a likelihood of meeting the MHC 9 class I binding requirements of the BALB/c (H-2d) and C57BL/6 (H-2b) haplotypes. Only 2 of 8 peptides 11 predicted by the algorithm and tested to date have 12 been identified as containing CTL epitopes. After in 13 vitro restimulation, the spleen cells were tested in a 14 standard Schromium-release assay well known in the art (see, for example, Hart et al. (1991) Proc. Natl.

16 Acad. Sci. USA 88: 9449-9452). Percent specific lysis 17 of peptide-coated, MHC-matched or mismatched target 18 cells was calculated as: 19 Experimental cpm- Spontaneous cpm x 100 21 Maximum cpm-Spontaneous cpm 22 23 Spontaneous cpm are the number of counts 24 released from target cells incubated in medium.

Maximum cpm are obtained by lysing target cells with 26 1% Triton X-100. Experimental cpm are the counts from 27 wells in which target cells are incubated with varying 28 numbers of effector (CTL) cells. Target cells tested 29 were L5178Y lymphoma or P815 mastocytoma cells (MHC matched to the H2 d BALB/c mice and EL4 lymphoma cells 31 (MHC matched to the H2b C57BL/6 mice). The 32 effector:target ratios tested were 25:1, 12:1, 33 6:1 and 3:1.

34 EXAMPLE 1 Survival Of Mice Inoculated With VRPs Encoding 36 Ebola Proteins. Mice were inoculated two or three 37 times at 1 month intervals with 2 x 106 focus-forming WO 00/00617 PCTIUS99/14311 1 units of VRPs encoding individual Ebola virus genes, 2 or Lassa virus NP as a control, or with phosphate 3 buffered saline (PBS). Mice were challenged with 4 pfu of mouse-adapted Ebola virus one month after the final immunization. The mice were observed daily, and 6 morbidity and mortality data are shown in Table 1A for 7 BALB/c mice and Table 1B for C57BL/6 mice. The viral 8 titers in individual sera of some mice on day 4 9 (BALB/c mice) or day 5 (C57BL/6 mice) following the initial viral challenge were determined by plaque 11 assay.

12 13 Table 1. Survival Of Mice Inoculated With VRPs Encoding Ebola Proteins A. BALB/c Mice VRP #Injections S/T 1 EboNP 3 30/3C 2 19/2C (100%) (95%) EboGP EboVP24 3 2 3 2 3 2 3 2 LassaNP 3 2 15/29 (52%) 14/20 (70%) 27/30 (90%) 19/20 (95%) 17/20 (85%) 11/20 (55%) 5/19 (26%) 4/20 (20%) 14/20 (70%) 17/20 (85%) 0/29 0/20

MDD

2 5/5 7 8 7 8 6 7 7 7 7 8 7 7 7 v v L.v treLL.mi 5.2 5/5 1/5 3/5 5/5 4/4 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 4.6 6.6 3.1 5.2 4.8 6.2 6.9 4.6 5.6 8.4 ST/m 3 u.rr~, WO 00/00617 WO 0000617PCTIUS99/1 4311 none (PBS) 3 2 1/30 0/20 5/5 5/5 8.3 8.7 3 4 B. C57BL/6 Mice 6 VRP #Iniections 1 (9 MD2 17-r, "4 =4 EboNP EboGP EboVP24 3 EboVP3O 3 3 EboVP4O 3 LassaNP 3 2 15/20 (75%) 8/10 (80%) 19/20 (95%) 10/10 (100%) 0/20 2/20 (10%) 14/20 (70%) 1/20 1/20 0/10 3/20 (15%) 0/10 10

ND

7 5/5 8 5/5 8 5/5 7 4/4 5/5 ND 4.1

ND

ND

8.6 7.7 7.8 8.6

ND

8.6

ND

4/4

ND

5/5

ND

none (PBS) 31 32 33 34 36 37 1 5/T, Survivors/total challenged.

2MDD, Mean day to death 3 V/T, Number of mice with viremia/total number tested.

4 Geometric mean of Log,, viremia titers in PFU/mL. Standard errors for all groups were 1.5 or less, except for the group of BALB/c mice given 2 inoculations of EboGP, which was 2.2.

'ND, not determined.

WO 00/00617 PCT[US99/1 4311 32 1 EXAMPLE 2 2 VP24-Immunized BALB/c Mice Survive A High-Dose 3 Challenge With Ebola virus.

4 BALB/c mice were inoculated two times with 2 x 106 focus-forming units of EboVP24VRP. Mice were 6 challenged with either 1 x 103 pfu or 1 x 10 5 pfu of 7 mouse-adapted Ebola virus 1 month after the second 8 inoculation. Morbidity and mortality data for these 9 mice are shown in Table 2.

11 Table 2. VP24-Immunized BALB/c Mice Survive A High- 12 Dose Challenge With Ebola virus 13 14 Replicon Challenge Dose Survivors/Total 16 EboVP24 1 x 103 pfu 17 (3 x 104 LD 5 0 18 19 EboVP24 1 x 105 pfu (3 x 106 LDs0 21 22 None 1 x 103 pfu 0/4 23 (3 x 10 4

LD

5 o) 24 None 1 x 105 pfu 0/3 26 (3 x 106 LDso) 27 28 29 EXAMPLE 3 31 Passive Transfer Of Immune Sera Can Protect 32 Naive Mice From A Lethal Challenge Of Ebola Virus.

33 Donor sera were obtained 28 days after the third 34 inoculation with 2 x 106 focus-forming units of VRPs encoding the indicated Ebola virus gene, the control 36 Lassa NP gene, or from unvaccinated control mice. One 37 mL of pooled donor sera was administered WO 00/00617 PCT/US99/1 4311 33 1 intraperitoneally (ip) to naive, syngeneic mice 24 h 2 prior to intraperitoneal challenge with 10 pfu of 3 mouse-adapted Ebola virus.

4 Table 3. Passive Transfer of Immune Sera Can Protect 6 Unvaccinated Mice from a Lethal Challenge of Ebola 7 Virus 8 9 A. BALB/c Mice Specificity of Survivors Mean Day 11 Donor Sera- /Total of Death 12 Ebola GP 15/20 8 13 Ebola NP 1/20 7 14 Ebola VP24 0/20 6 Ebola VP30 0/20 7 16 Ebola VP35 ND 1

ND

17 Ebola VP40 0/20 6 18 Lassa NP 0/20 7 19 Normal mouse sera 0/20 6 21 B. C57BL/6 Mice 22 Specificity of Survivors Mean Day 23 Donor Sera- /Total of Death 24 Ebola GP 17/20 7 Ebola NP 0/20 7 26 Ebola VP24 ND ND 27 Ebola VP30 ND ND 28 Ebola VP35 0/20 7 29 Ebola VP40 ND ND Lassa NP 0/20 7 31 Normal mouse sera 0/20 7 32 33 1 ND, not determined 34 36 37 WO 00/00617 PCT/US99/14311 34 1 EXAMPLE 4 2 Immunoqenicitv and Efficacy of VReDEboGP and 3 VRepEboNP in Guinea Pigs.

4 EboGPVRP or EboNPVRP (1x10 7 IU in 0.5ml PBS) were administered subcutaneously to inbred strain 2 or 6 strain 13 guinea pigs (300-400g). Groups of five 7 guinea pigs were inoculated on days 0 and 28 at one 8 (strain 2) or two (strain 13) dorsal sites. Strain 13 9 guinea pigs were also boosted on day 126. One group of Strain 13 guinea pigs was vaccinated with both the 11 GP and NP constructs. Blood samples were obtained 12 after vaccination and after viral challenge.

13 Sera from vaccinated animals were assayed for 14 antibodies to Ebola by plaque-reduction neutralization, and ELISA. Vaccination with VRepEboGP 16 or NP induced high titers of antibodies to the Ebola 17 proteins (Table 4) in both guinea pig strains.

18 Neutralizing antibody responses were only detected in 19 animals vaccinated with the GP construct (Table 4).

Guinea pigs were challenged on day 56 (strain 2) 21 or day 160 (strain 13) by subcutaneous administration 22 of 1000 LD 50 (104 PFU) of guinea pig-adapted Ebola 23 virus. Animals were observed daily for 60 days, and 24 morbidity (determined as changes in behavior, appearance, and weight) and survival were recorded.

26 Blood samples were taken on the days indicated after 27 challenge and viremia levels were determined by plaque 28 assay. Strain 13 guinea pigs vaccinated with the GP 29 construct, alone or in combination with NP, survived lethal Ebola challenge (Table Likewise, 31 vaccination of strain 2 inbred guinea pigs with the GP 32 construct protected 3/5 animals against death from 33 lethal Ebola challenge, and significantly prolonged 34 the mean day of death (MDD) in one of the two animals that died (Table Vaccination with NP alone did 36 not protect either guinea pig strain.

WO 00/00617 PCTIUS99/1 4311 Table 4. Immunogenicity and efficacy of VRepEboGP and VRepEboNP in guinea pigs A. Strain 2 guinea pigs Survivors/ Viremiac VRP ELISAa PRNT total(MDDb) d7 d14 GP 4.1 30 3/5 (13+2.8) 2.3 1.8 NP 3.9 <10 0/5 3.0 Mock <1.5 <10 0/5 3.9 B. Strain 13 guinea pigs Survivors/ Viremiac VRP ELISAa PRNT, 0 total (MDDb) d7 d14 GP 4.0 140 5/5 <2.0 GP/NP 3.8 70 5/5 <2.0 NP 2.8 <10 1/5(8.3+2.2) 4.6 Lassa NP <1.5 <10 2/5(8.3+0.6) 4.8 aData are expressed as geometric mean titers, log, 0 bMDD, mean day to death 'Geometric mean of log, 1 viremia titers in PFU/mL. Standard errors for all groups were 0.9 or less.

EXAMPLE Induction of murine CTL responses to Ebola virus NP and Ebola virus VP24 proteins.

BALB/c and C57BL/6 mice were inoculated with VRPs encoding Ebola virus NP or VP24. Mice were euthanized at various times after the last inoculation and their spleens removed. Spleen cells (1 x 106/ ml) were cultured in vitro for 2 days in the presence of to 25 pM of peptides, and then for an additional days in the presence of peptide and 10% supernatant from concanavalin A-stimulated syngeneic spleen cells.

After in vitro restimulation, the spleen cells were tested in a standard "Schromium-release assay. Percent specific lysis of peptide-coated, MHC-matched or mismatched target cells was calculated as: WO 00/00617 PCT/US99/14311 Experimental cpm- Spontaneous cpm x 100 Maximum cpm-Spontaneous cpm In the experiments shown, spontaneous release did not exceed Table 5. Induction of murine CTL responses to Ebola virus NP and Ebola virus VP24 proteins.

Mice,VRP 1 BALB/c,VP24 BALB/c,VP24 C57BL/6,EboNP C57BL/6,EboNP 4 C57BL/6,EboNP C57BL/6,LassaNP C57BL/6,LassaNP C57BL/6,LassaNP C57BL/6,LassaNP Peptide 2 None SEQ ID I None SEQ ID I Lassa NI None SEQ ID I None SEQ ID I Specific Lysis E:T ratio Cell 3 P815 NO:25 P815 93 EL4 2 NO:24 EL4 SEL4 2 L5178Y 1 O:24 L5178Y 0 EL4 2 1O:24 EL4 6 1 Indicates the mouse strain used and the VRP used as the in vivo immunogen. In vitro restimulation was performed using SEQ ID NO:24 peptide for BALB/c mice and SEQ ID NO:23 for all C57BL/6 mice shown.

2 Indicates the peptide used to coat the target cells for the chromium release assay.

3 Target cells are MHC-matched to the effector cells, except for the L5178Y cells that are C57BL/6 mismatched.

4 High levels of specific lysis were also observed using E:T ratios of 12, 6, 3, or 1:1.

RESULTS AND DISCUSSION Ebola Zaire 1976 (Mayinga) virus causes acute hemorrhagic fever characterized by high mortality.

There are no current vaccines or effective therapeutic measures to protect individuals who are exposed to this virus. In addition, it is not known which genes WO 00/00617 PCT/US99/1 4311 37 1 are essential for evoking protective immunity and 2 should therefore be included in a vaccine designed for 3 human use. In this study, the GP, NP, VP24, 4 VP35, and VP40 virion protein genes of the Ebola Zaire 1976 (Mayinga) virus were cloned and inserted into a 6 Venezuelan equine encephalitis (VEE) virus replicon 7 vector (VRep) as shown in Figure 2A and 2B. These 8 VReps were packaged as VEE replicon particles (VRPs) 9 using the VEE virus structural proteins provided as helper RNAs, as shown in Figure 3. This enables 11 expression of the Ebola virus proteins in host cells.

12 The Ebola virus proteins produced from these 13 constructs were characterized in vitro and were shown 14 to react with polyclonal rabbit anti-Ebola virus antibodies bound to Protein A beads following SDS gel 16 electrophoresis of immunoprecipitated proteins (Figure 17 4).

18 The Ebola virus genes were sequenced from the VEE 19 replicon clones and are listed here as SEQ ID NO:1 2 3 (VP24), 4 (VP30), 5 (VP35), 6 21 and 7 (VP30#2) as described below. The corresponding 22 amino acid sequences of the Ebola proteins expressed 23 from these replicons are listed as SEQ ID NO: 17, 18, 24 19, 20, 21, 22, and 23, respectively. Changes in the DNA sequence relative to the sequence published by 26 Sanchez et al. (1993) are described relative to the 27 nucleotide (nt) sequence number from GenBank 28 (accession number L11365).

29 The sequence we obtained for Ebola virus GP (SEQ ID NO:1) differed from the GenBank sequence by a 31 transition from A to G at nt 8023. This resulted in a 32 change in the amino acid sequence from Ile to Val at 33 position 662 (SEQ ID NO: 17).

34 The DNA sequence we obtained for Ebola virus NP (SEQ ID NO:2) differed from the GenBank sequence at 36 the following 4 positions: insertion of a C residue 37 between nt 973 and 974, deletion of a G residue at nt 38 979, transition from C to T at nt 1307, and a WO 00/00617 PCT/US99/14311 38 I transversion from A to C at nt 2745. These changes 2 resulted in a change in the protein sequence from Arg 3 to Glu at position 170 and a change from Leu to Phe at 4 position 280 (SEQ ID NO: 18).

The Ebola virus VP24 (SEQ ID NO:3) gene differed 6 from the GenBank sequence at 6 positions, resulting in 7 3 nonconservative changes in the amino acid sequence.

8 The changes in the DNA sequence of VP24 consisted of a 9 transversion from G to C at nt 10795, a transversion from C to G at nt 10796, a transversion from T to A at 11 nt 10846, a transversion from A to T at nt 10847, a 12 transversion from C to G at nt 11040, and a 13 transversion from C to G at nt 11041. The changes in 14 the amino acid sequence of VP24 consisted of a Cys to Ser change at position 151, a Leu to His change at 16 position 168, and a Pro to Gly change at position 233 17 (SEQ ID NO: 19).

18 We have included 2 different sequences for the 19 Ebola virus VP30 gene (SEQ ID NOS:4 and SEQ ID NO:7).

Both of these sequences differ from the GenBank 21 sequence by the insertion of an A residue in the 22 upstream noncoding sequence between nt 8469 and 8470 23 and an insertion of a T residue between nt 9275 and 24 9276 that results in a change in the open reading frame of VP30 and VP30#2 after position 255 (SEQ ID 26 NOS:20 and SEQ ID NO:23). As a result, the C-terminus 27 of the VP30 protein differs significantly from that 28 previously reported. In addition to these 2 changes, 29 the VP30#2 gene in SEQ ID NO:23 contains a conservative transition from T to C at nt 9217.

31 Because the primers originally used to clone the 32 gene into the replicon were designed based on the 33 GenBank sequence, the first clone that we constructed 34 (SEQ ID NO:4) did not contain what we believe to be the authentic C-terminus of the protein. Therefore, 36 in the absence of the VP30 stop codon, the C-terminal 37 codon was replaced with 37 amino acids derived from 38 the vector sequence. The resulting VP30 construct WO 00/00617 PCT/US99/1 4311 39 1 therefore differed from the GenBank sequence in that 2 it contained 32 amino acids of VP30 sequence 3 (positions 256 to 287, SEQ ID NO:20) and 37 amino 4 acids of irrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place of the C-terminal 5 amino 6 acids reported in GenBank. However, inclusion of 37 7 amino acids of vector sequence in place of the C- 8 terminal amino acid (Pro, SEQ ID NO:23) did not 9 inhibit the ability of the protein to serve as a protective antigen in BALB/c mice. We are currently 11 examining the ability of the new VEE replicon 12 construct (SEQ ID NO:7), which we believe contains the 13 authentic C-terminus of VP30 (VP30#2, SEQ ID NO:23), 14 to protect mice against a lethal Ebola challenge.

The DNA sequence for Ebola virus VP35 (SEQ ID 16 NO:5) differed from the GenBank sequence by a 17 transition from T to C at nt 4006, a transition from T 18 to C at nt 4025, and an insertion of a T residue 19 between nt 4102 and 4103. These sequence changes resulted in a change from a Ser to a Pro at position 21 293 and a change from Phe to Ser at position 299 (SEQ 22 ID NO:21). The insertion of the T residue resulted in 23 a change in the open reading frame of VP35 from that 24 previously reported by Sanchez et al. (1993) following amino acid number 324. As a result, Ebola virus 26 encodes for a protein of 340 amino acids, where amino 27 acids 325 to 340 (SEQ ID NO:21) differ from and 28 replace the C-terminal 27 amino acids of the 29 previously published sequence.

Sequencing of VP30 and VP35 was also performed 31 on RT/PCR products from RNA derived from cells that 32 were infected with Ebola virus 1976, Ebola virus 1995 33 or the mouse-adapted Ebola virus. The changes noted 34 above for the VRep constructs were also found in these Ebola viruses. Thus, we believe that these changes are 36 real events and not artifacts of cloning.

37 The Ebola virus VP40 differed from the GenBank 38 sequence by a transversion from a C to G at nt 4451 WO 00/00617 PCT/US99/14311 1 and a transition from a G to A at nt 5081. These 2 sequence changes did not alter the protein sequence of 3 VP40 (SEQ ID NO:22) from that of the published 4 sequence.

To evaluate the protective efficacy of 6 individual Ebola virus proteins and to determine 7 whether the major histocompatibility (MHC) genes 8 influence the immune response to Ebola virus antigens, 9 two MHC-incompatible strains of mice were vaccinated with VRPs expressing an Ebola protein. As controls for 11 these experiments, some mice were injected with VRPs 12 expressing the nucleoprotein of Lassa virus or were 13 injected with phosphate-buffered saline (PBS).

14 Following Ebola virus challenge, the mice were monitored for morbidity and mortality, and the results 16 are shown in Table 1.

17 The GP, NP, VP24, VP30, and VP40 proteins of 18 Ebola virus generated either full or partial 19 protection in BALB/c mice, and may therefore be beneficial components of a vaccine designed for human 21 use. Vaccination with VRPs encoding the NP protein 22 afforded the best protection. In this case, 100% of 23 the mice were protected after three inoculations and 24 95% of the mice were protected after two inoculations.

The VRP encoding VP24 also protected 90% to 95% of 26 BALB/c mice against Ebola virus challenge. In separate 27 experiments (Table two or three inoculations with 28 VRPs encoding the VP24 protein protected BALB/c mice 29 from a high dose (1 x 10 5 plaque-forming units (3 x 106 LD50)) of mouse-adapted Ebola virus.

31 Vaccination with VRPs encoding GP protected 52- 32 70% of BALB/c mice. The lack of protection was not 33 due to a failure to respond to the VRP encoding GP, as 34 all mice had detectable Ebola virus-specific serum antibodies after vaccination.

36 Some protective efficacy was also observed in 37 BALB/c mice vaccinated two or three times with VRPs 38 expressing the VP30 protein (55% and WO 00/00617 PCT/US99/14311 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 33 34 36 37 38 respectively),or the VP40 protein (70% and respectively). The VP35 protein was not efficacious in the BALB/c mouse model, as only 20% and 26% of the mice were protected after either two or three doses, respectively.

Geometric mean titers of viremia were markedly reduced in BALB/c mice vaccinated with VRPs encoding Ebola virus proteins after challenge with Ebola virus, indicating an ability of the induced immune responses to reduce virus replication (Table 1A). In this study, immune responses to the GP protein were able to clear the virus to undetectable levels within 4 days after challenge in some mice.

When the same replicons were examined for their ability to protect C57BL/6 mice from a lethal challenge of Ebola virus, only the GP, NP, and proteins were efficacious (Table 1B). The best protection, 95% to 100%, was observed in C57BL/6 mice inoculated with VRPs encoding the GP protein.

Vaccination with VRPs expressing NP protected 75% to 80% of the mice from lethal disease. In contrast to what was observed in the BALB/c mice, the VP35 protein was the only VP protein able to significantly protect the C57BL/6 mice. In this case, 3 inoculations with VRPs encoding VP35 protected 70% of the mice from Ebola virus challenge. The reason behind the differences in protection in the two mouse strains is not known but is believed to be due to the ability of the immunogens to sufficiently stimulate the cellular immune system. As with the BALB/c mice, the effects of the induced immune responses were also observed in reduced viremias and, occasionally, in a prolonged time to death of C57BL/6 mice.

VRPs expressing Ebola virus GP or NP were also evaluated for protective efficacy in a guinea pig model. Sera from vaccinated animals were assayed for antibodies to Ebola by western blotting, IFA, plaquereduction neutralization, and ELISA. Vaccination with WO 00/00617 PCT/US99/14311 42 1 either VRP (GP or NP) induced high titers of 2 antibodies to the Ebola proteins (Table 4) in both 3 guinea pig strains. Neutralizing antibody responses 4 were only detected in animals vaccinated with the VRP expressing GP (Table 4).

6 Vaccination of strain 2 inbred guinea pigs with 7 the GP construct protected 3/5 animals against death 8 from lethal Ebola challenge, and significantly 9 prolonged the mean day of death in one of the two animals that died (Table All of the strain 13 11 guinea pigs vaccinated with the GP construct, alone or 12 in combination with NP, survived lethal Ebola 13 challenge (Table Vaccination with NP alone did not 14 protect either guinea pig strain from challenge with the guinea pig-adapted Ebola virus.

16 To identify the immune mechanisms that mediate 17 protection against Ebola virus and to determine 18 whether antibodies are sufficient to protect against 19 lethal disease, passive transfer studies were performed. One mL of immune sera, obtained from mice 21 previously vaccinated with one of the Ebola virus 22 VRPs, was passively administered to unvaccinated mice 23 24 hours before challenge with a lethal dose of mouse- 24 adapted Ebola virus. Antibodies to GP, but not to NP or the VP proteins, protected mice from an Ebola virus 26 challenge (Table Antibodies to GP protected 75% of 27 the BALB/c mice and 85% of the C57BL/6 mice from 28 death. When the donor sera were examined for their 29 ability to neutralize Ebola virus in a plaquereduction neutralization assay, a 1:20 to 1:40 31 dilution of the GP-specific antisera reduced the 32 number of viral plaque-forming units by at least 33 (data not shown). In contrast, antisera to the NP and 34 VP proteins did not neutralize Ebola virus at a 1:20 or 1:40 dilution. These results are consistent with 36 the finding that GP is the only viral protein found on 37 the surface of Ebola virus, and is likely to induce 38 virus-neutralizing antibodies.

WO 00/00617 PCT/US99/14311 43 1 Since the NP and VP proteins of Ebola virus are 2 internal virion proteins to which antibodies are not 3 sufficient for protection, it is likely that cytotoxic 4 T lymphocytes (CTLs) are also important for protection against Ebola virus. Initial studies aimed at 6 identifying cellular immune responses to individual 7 Ebola virus proteins expressed from VRPs identified 8 CTL responses to the VP24 and NP proteins (Table 9 One CTL epitope that we identified for the Ebola virus NP is recognized by C57BL/6 (H-2 b) mice, and has an 11 amino acid sequence of, or contained within, the 12 following 11 amino acids: VYQVNNLEEIC (SEQ ID NO:24).

13 Vaccination with EboNPVRP and in vitro restimulation 14 of spleen cells with this peptide consistently induces strong CTL responses in C57BL/6 (H-2b) mice. In vivo 16 vaccination to Ebola virus NP is required to detect 17 the CTL activity, as evidenced by the failure of cells 18 from C57BL/6 mice vaccinated with Lassa NP to develop 19 lytic activity to peptide (SEQ ID NO:24.) after in vitro restimulation with it. Specific lysis has been 21 observed using very low effector:target ratios 22 This CTL epitope is H-2b restricted in that it is not 23 recognized by BALB/c (H-2d) cells treated the same way 24 (data not shown), and H-2b effector cells will not lyse MHC-mismatched target cells coated with this 26 peptide.

27 A CTL epitope in the VP24 protein was also 28 identified. It is recognized by BALB/c (H-2d) mice, 29 and has an amino acid sequence of, or contained within, the following 23 amino acids: 31 LKFINKLDALLVVNYNGLLSSIF (SEQ ID NO:25). In the data 32 shown in Table 5, high specific lysis of P815 33 target cells coated with this peptide was observed.

34 The background lysis of cells that were not peptidecoated was also high which is probably due to 36 the activity of natural killer cells. We are planning 37 to repeat this experiment using the L5178Y target WO 00/00617 PCT/US99/14311 44 1 cells, which are not susceptible to natural killer 2 cells.

3 Future studies will focus on determining the 4 fine specificities of these CTL responses and the essential amino acids that constitute these CTL 6 epitopes. Additional studies to identify other CTL 7 epitopes on Ebola virus GP, NP, VP24, VP30, VP35, and 8 VP40 will be performed. To evaluate the role of these 9 CTLs in protection against Ebola virus, lymphocytes will be restimulated in vitro with peptides containing 11 the CTL epitopes, and adoptively transferred into 12 unvaccinated mice prior to Ebola virus challenge. In 13 addition, future studies will examine the CTL 14 responses to the other Ebola virus proteins to better define the roles of the cell mediated immune responses 16 involved in protection against Ebola virus infection.

17 18 19 21 22 23 24 26 27 28 29 31 32 33 34 36 37 EDITORIAL NOTE APPLICATION NUMBER 50844/99 The following Sequence Listing pages 1-24 are part of the description. The claims pages follow on pages 45-51.

WO 00/00617 PCT/US99/14311 1 SEQUENCE LISTING <110> United States Army Medical Research Institute of Infectious Diseases Hart, Mary Katherine Wilson, Julie A.

Pushko, Peter Smith, Jonathan F.

Schmaljohn, Alan L.

<120> Ebola Virion Proteins Expressed from Venezuelan Equine Encephalitis (VEE) Virus Replicons <130> 003/141/SAP <140> PCT/US99/14311 <141> 1999-06-22 <150> US 60/091,403 <151> 1998-06-29 <160> <170> Apple Macintosh Microsoft Word <210> 1 <211> 2298 <212> DNA <213> Ebola Zaire <220> <400> 1 atcgataagc tcggaattcg agctcgcccg gggatcctct agagtcgaca acaacacaat gggcgttaca ggaatattgc agttacctcg tgatcgattc aagaggacat cattctttct 120 ttgggtaatt atccttttcc aaagaacatt ttccatccca 160 cttggagtca tccacaatag cacattacag gttagtgatg 200 tcgacaaact agtttgtcgt gacaaactgt catccacaaa 240 tcaattgaga tcagttggac tgaatctcga agggaatgga 280 gtggcaactg acgtgccatc tgcaactaaa agatggggct 320 tcaggtccgg tgtcccacca aaggtggtca attatgaagc 360 tggtgaatgg gctgaaaact gctacaatct tgaaatcaaa 400 SUBSTITUTE SHEET (RULE 26) WO 00/00617 WO 0000617PCT/US99/I 4311 aaacc tgacc ggat tcggg5 atcaggaacc aaagagggtc cagttatcta tgcatttctc agctcacacc acccgtctac ggctaccggt gaggttgaca tcacaccaca tacaagtggg tggaaggtca gggccttctg tcgcagtgaa gc caaaaaca ccgacccagg catggcttca agtcaaggaa ttgccacaat accaggtccg aaacttgaca accgcagaac ctctgccacg accaacacga ccacaacaag caacaacac t agcagcggga gagtcgcagg agaagcaat t ttacattact gactggcctg aatttacata atctgtgggt ctcttcaac t cttttcaatc cagcgatggg gctgtatcga caaaattgat c ttccggacc ggagacaatg tgtaattgca gtcttttagt agcctcaaat acttgaatct actggagctt taaactcaca gcagccaagc

L

L

t q 9 t

C

a 9 9 t ggagtgagtg tctaccagca cttcccccgg tgccggtatg ggaccgtgtg ccggagactt ctttcttcct gtatgatcga ccgaggaacg actttcgctg atactgcccc aagctaagaa ccttgagaga gccggtcaat tggctactat tctaccacaa t ttggaacca atgagacaga atttgaccta cgtccaactt gtttctgctc cagctgaatg aaaaggagca ataccacggg accccgaaat tgatacaaca ggaaactaaa aaaaacctca gagttgtctt tcacagttgt tcagtggtca gagtccggcg gaccaacaca acaactgaag gaaaattcct ctgcaatggt gggaagctgc agtgtcgcat ctccacgagt ccccaatccc gacaacagca cccataatac tctctgaggc aactcaagtt agacaacgac agcacagcc t accgcagccg gacccccaaa jcaagagcac tgacttcctg :ccccaaaac cacagcgaga :atcaccaag ataccggaga igctaggctt aattaccaat ictgatcaca ggcgggagaa Itcaatgctc aacccaaatg lgactactca ggatgaaggt lataccatat ttcgggccag ;aggggctaa tgcacaatca :gagacagct ggccaacgag ;ttcctgaga gccacaactg :tcaaccgta aggcaattga ~cggcacatg ccacattctg Lccacatgat tggaccaaga :agattattc atgattttgtI ~gggggacaa tgacaattgg1 rataccggca ggtattggag rttatcgctt tattctgtat .tttcttcag attgcttcat aatgaaacc aggatttaat .agattactt gacaaatgat z .aaacatagc caatgtgatt c ttaatcata aacaaggttt E gcgccagacg tgcacaaagt tgccttccat cttgcttcca aaggtgtcgt ggac ttc ttc gcaacggagg ttagatatca gtacttgttc gaatcaagat agacaatata aaaactaatt atcggggagt ctagaaaaat atcaaacgga cgaacttctt accacaaaat tcaagtgcac c taacaaccc tcacaaccaa acccgtgtat gaacaagatc ccgacactcc agcagagaac gaccccgcca ccgctggcaa agagagtgcc actattgctg gaac tcgaag caaccc taat gctgcaatcg cagc cgaggg agatggttta acgactcaag agctacgcac tttcttgctg lgaccggac t acataacaga tgataaaacc :ggacaggat :tacaggcgt 3.tgcaaattt lgaaaagc tc :atatggatt Latataatac :taactcctt ~agtcgacct 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000 1040 1080 1120 1160 1200 1240 1280 1320 1360 1400 1440 1480 1520 1560 1600 1640 1680 1720 1760 1800 1840 1880 1920 1960 2000 2040 2080 2120 2160 2200 2240 2280 2298 tatcgat <210> 2 <211> 2428 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/1 4311 <212> DNA <213> Ebola Zaire <220> <400> 2 atcgataagc gtatggattc tctcactgaz gcaggtctgt tcatcccagt ccaacttatc caagagagtc atcatgcgta tggcgcagtc gaagtcaaga tgctgccagc acttgctgcc gccggtcagt aattggtagt aaggcaaatt tatccaacag ttttccgttt cctaatacac gccaacgatg gt ttttcagg tatcc tacaa cttgcaagga ttaaggctgc tgctcctttc aatcttgagc cactcggagt agtaaatgtt gccac tgagg ctcgcgaact gaaaattctt agcttccagc agcgc ctggc actgcccaaa attccctttc gccatcaaga cattcccgat ggcgaatacc cagatgactt ttggctgcag tcgtcctcag i tctgacatgg :ccgttcaaca :gtatcaagta "atacaggcct Tcggacagttt Lccagggagat aagtatttgg agcgtgatgg agtatctagt atgccggaag ttctctcctt aggagaaaag caagtacatg cttggcaatc gatgcgaaca caagggatgc ctgtgatttc cttattgatt aagacagaac ccgccaaggt actcagctcc gcccgacttt atggtctttt cgccacagca ggagaacagt ctgagaagca tgaccatctt atgaacttcc aaacaaacgc caagctgaca acaagtggac caggacccat tgatgatccg gtggtggttg a agagttactc E ggtcctattc c gtcgactcta aaaatc tgga attaccacaa ggggattgtt aacaatcttg t tgaagcagg ccttctgatg tacaaacttt aagggcacgg agtgaagcga ggaaaaaaca aggagacaac tgcaagtc ta gcttgccttg cagagc aagg agtaggacac aattttctga acatggttgc aaattcagtg gtcaaaacag gaggagttcg aaaaaatgag ctggccaagc tgaacctttc1 ccctcaacta cacgggagta atcaacaact actccaacaa ggacttgatg atcagaaaaa tatggtaact jaagctatca attacgatgat -aatgatgac ictgactcac F ltcccgatga t ;gaaaacggc a ~atctagacg a %tagatcgac c Iggccagcat a ittcaaaatg t :cagtgcgcc a :tccggctca a raagaggcag a tagccttcc g agggacgga a gaggatccga tggcgccgag gatcttgaca 120 cggcaaagag 160 aagaaatttg 200 tgttgatttt 240 ctttgtcttc 280 tcttggaaag 320 gttccgtttt 360 cttgaggaat 400 ttaagagaac 440 tgaagctaat 480 ttccttccga 520 agaaggttca 560 actgatacaa 600 atgatggtga 640 tcaaatttct 680 cgggcatgat 720 gctcaagctc 760 tacttgatca 800 tctccatcct 840 gtgaactcct 880 atggagagta 920 tggagtaaat 960 tcggcaattg 1000 ccctcgcagg 1040 cagagaggct 1080 :atgcagagt 1120 itcaggaaaa 1160 laacgaaatc 1200 :taagaaaag 1240 :tgctgcgtc 1280 :gatgacgac 1320 ~acaatccta 1360 ~ggatacgac 1400 :ggaagctac 1440 tgaatgcac 1480 iggacgacga 1520 :aagggtgga 1560 ~tagagggca 1600 .cccaggccc 1640 .ctcacggac 1680 .ccagccctc 1720 cccactgga 1760 cccttggag 1800 cttccaacc 1840 ggacactaag ccagtgccta caacagaaga acagtcaaaa gacagacaca atccaggcca tcacagaaca atccaccacg aatgacagaa gaaatgaacc gcatgctgac accaattaac cgatgccgac gacgagacgt tcagatgatg aagagcagga SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/1 4311 gcacacccac tgtcgcccca tcactctgaa aagaaagaac ccggctcccg tatacagaga gatcaggacc acaacaccca ccacattcta tattatcata ccagtgatgg agaggaatat aa tgaagaga t ttat tggcc aatcctgcaa gatgattcaa gaacgaaaac ttattatcac gcgagctcga acac tcaaga gtcagaacac agatcacagg tgatgaagga caaagagtac ccaccatggc atagatttgt ggtgatgaat catcatcagt ccgacaaata aggaagaatt aataaaagtg attcccgagc tcccgcaaga ggccaggaac tcttttgagg ggccatttga tgagcctgta acgtatccag tcactgaaaa tacattggat cacaagaata gaatgagcat gctaacatta tttgatgtct attcttattt ttatcgat cgagcaacaa caggacagtg agatgtatcg tgctgttttg gttttcagta actcccttga agaggctatg ggtcaacaat aattcatggc ggaacaatgg agtagtccag aaggtgtgaa ttgaatttgg 1880 1920 1960 2000 2040 2080 2120 2160 2200 2240 2280 2320 2360 2400 2428 <210> 3 <211> 847 <212> DNA <213> Ebola Zaire <220> <400> 3 atcgatctcc tggc taaagc aaaggac ctg aacttcttag attgggctgg ggccctattg gcatggtcaa aaaatccgaa gagagtcatc gaccagtctt tgatctctga caacatgcga aaaatgc tgt ttaacaaatt attgttgagc atcatcataa tccaagaacc tgggccggcg aaagcattta tg'attcttga gtagaatac t catcgat agacaccaag tacgggacga gagaaagggg ttagccaaac tattgagttt catagactga tgacaaggaa ttccacaatt cttgcagcag tgattgaacc ttggctgcta acacaacgtg cgttgattcg ggatgctcta agtattgaaa ctcgaactaa cgacaaatcg aaattttccc cacaaggatc atttaatagc tcatattgag caagac ctga tacaatctaa ttgtcttaag tattcagggg gatgtgactc aaactaatga tctctttcct gaatcaccgc ggatacagga c ttagcagga acaaccaaca tcaaggaaca atccaatat t catgtcgtga ttggaactca catgggtttt gcaatgaacc tcc ttcatga ctcgacacga tctcttgcta ctaactcata gaaaaaacca tatcgcccaa cgacctctgt tggaaggt tt acaaaggaat ctttgcccct catttatttc tgtgggcatt ccagctgatt gcccttggtc ctaaccattt attgagccta ctcaagttta actacaacgg aaatcataca c tggtggagc gcatgaagcc gtccacactg atgcaaagtt tc taactaag tatgctgact 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 847 <210> 4 SUBSTITUTE SHEET (RULE 26) WO 00/00617 WO 0000617PCTIUS99/1 4311 <211> 973 <212> DNA <213> Ebola Zaire <220> <400> 4 atcgatcaga taacacacat taaacagtga atgagagagg ggatggacac agagagaat t cc tcacaagt agttgaacca tgtccgacct tttgcaaaaa ggaat tactc gtagaacaac gcttagcaaa aggtccaaaa gaacactggg caaaattaag gaggaaattc acacacctaa aacccgttct aggaggcagt ctacaatccc ttgctctcca ttcagggtta gaagcttcaa agggtacatc tctgcgaacc aaagcat tgg gtggagacaa acgcccacga gaccaccatg atcgaggtga gcgcgttcct ttaacagttc tgaaaaaagg agatcaccag ctactaatcg aattaaatat tccaacggct attaccttgt cgagacaaga agcattgttg tcaaaatccc ggcgcgaggg cgaagtatat tttgaagctg taattatgtt gttaccgtgt agaacattgg ccaacccggg gat ggtagagt tt tcaaaaagtc cttttaaatg gc tgccagac ttcgagcacg gtaccgtcaa actgtatttc ctccagcacc atttttgtgt ttggagagt t cccgtaagac aactgcaccc gatgatttcc tgacactgat catcagaacc actctatgtg agc tgagtc t gc ttgggcaa caacgattac cactatggca tatcactgca gaaagttctg ttcctcaatc gacatgctca agttgcaacc aatagaaatt gaagcttcat agcattcaag atcatcatcc tcaaggagcg ataagaagag taaagacata gacagtagtt taactgatag t tgtggatca aaggac tcgc agcaagagga caagacggca atagaggatt c tgtgatgac tttatgtgag gatcaggcag acagtgataa acaatgggac t tc ttgaata c tgtcgttgt agataatgag tggtctgatg 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800- 840 880 920 960 973 <210> <211> 1148 <212> DNA <213> Ebola Zaire <220> <400> atcgatagaa aagctggtct aacaagatga caactagaac aaagggcagg ggccatactg cggccacgac tcaaaacgac agaatgccag gccctgagct ttcgggctgg atctctgagc agctaatgac cggaagaatt cctgtaagcg acatcttctg tgatattgag aacaatccag gattatgcta cgcatcccaa atgcaacaaa cgaagccaaa cccgaagacg cgcaacagtc 120 160 200 240 SUBSTITUTE SHEET (RULE 26) WO 00/00617 WO 0000617PCT/US99/1 4311 aaacccaaac ggtagtacaa caacaaacca cgagtcttga aaaaacaatc gttgcaaaat caacagcaac acatggtcaa agtgcgattc tccctcaaag taccacttca atttcggcaa tgcctggtt t gatttgtaaa attcatgctg ctcctcaatg aatcttccaa tctcgaggtg gtccagtccc atgtgttttt aaaatttgag taatagcaga acattaatga ggacccaatt acattggctt tcgcatcaga gaatggtcta tcctcattga atgatc ttct cgctgcggca ccaccacctg ggggtaagat tgttagggag c taac tgagg aggatttgag tggaac tgc t t tgggaaaag agttccaggc tgccctaatt gatgctgctc acattccccg accatcgccc cagc ttcaag ccaatctccc ggcttcaact tacacttgtg tgcaatcata gttttgagga cattggctac tgttgtgcaa atcattagaa caacgcatta aagccagttt atgatatggc acagggtttg tgctgagatg ggtgatgaca accggtcggg actgaggctt attgggccga gaccatcact ttatgaagaa tgaatctaga gatgagaccg gcattcaaca atctaaacag aaaattttgg gaaacctgac aaacattatg tatgatcact ttccaccaat tagtacaagt atagcaactc attggacatc cagcctggct gaaggagac t caaattacaa aaagagttcc cacctgtcat ccacatccgc agcttgccag aaaagcttgc aagattgatc gaggttgggt atggtaaaac acttggactc ttccctccga aagaggcgaa gctgaactat agggtacgtt agatcgat 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000 1040 1080 1120 1148 <210> 6 <211> 1123 <212> DNA <213> Ebola Zaire <22 0> <400> 6 atcgatccta cctcggctga cttcatcttg taaacgttga aggcgggtta tattgcctac aggccatata ccctgtcagg aggtggcaac agcaatacag gtcaatgggg acactccatc ccgatgacac catcgaccat tgtgtcatca gcattcatcc atatcgggcc ccaaagtgct ggcttcctct aggtgtcgct tgactcaact acggccgcca atcacccatt tcggcaaggc tcaatcggct gggtcctgga gctcctgcga attggaaacc gttcttccgc cagtccaact atttgacagc actcaaactg tgcaacatgg accgatgaca gagagtgttt tttcattaac gcaaaattgt taaaaatatg tgctcctcct gaatatatgg tcaaattcaa caattgctag gcttcctgac accggagtca gaatccactc aggccaattg gccagccaca caccaggcag ttgaagctat ggtgaatgtc aatgaagcaa attccaattt gatcaaaaga cctacagctt tcatgcttgc ttcatacact aaccaatcca cttgtcagag atcccggatc atcccctcag aggctttcct ccaggagttc accccagtat ttcacctttg atcacccaac cactgcctgc ctccaacagg atcaaatgga 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 SUBSTITUTE SHEET (RULE 26) WO 00/00617 WO 0000617PCTJUS99/1 4311 gcgt tgcgtc ccattctttt tgccgatcta acttcactcc ccaaaaatat ccacaagctg caaccaatca tggacccggt acaggattgt gctgtgattg agttttatag gat caggaatttc acccaacaaa acatc tc cgg aggactttaa catgggaatc accggtaaga tccctgttct ggc tccagga gacacgtgtc agaagtaat t aatcttctca atttcatcca agtgggaaga agaaaatcca gatcgttcca gaagtgccag aggtgacttc tttgccaaag gacctcacca attc tc ctgc gcaataattg gggatagtgc aaacttcgcc aggggaacag agcaataatg attgatccaa aaac tctggt taaaaatgga tacattgggt tggtaatcac aagtcttcca ac tcagatcc ataacatatc 720 760 800 840 880 920 960 1000 1040 1080 1120 1123 <210> 7 <211> 1165 <212> DNA <213> Ebola Zaire <220> <400> 7 atcgatcaga taacacacat taaacagtga atgagagagg ggatggacac agagagaatt cctcacaagt agttgaacca tgtccgacct tttgcaaaaa ggaattactc gtagaacaac gcttagcaaa aggtccaaaa gaacactggg caaaattaag gaggaaattc acacacc taa aacccgttc t aggaggcagt cgacaatccc ttgctctcca ttcagggtta gaagc ttcaa agggtacccc cttctacttg tatttaatca tataatcac t tgccc taata tctgcgaacc aaagcattgg gtggagacaa acgcccacga gaccaccatg atcgaggtga gcgcgttcct ttaacagttc tgaaaaaagg agatcaccag ctactaatcg aattaaatat tccaacggct attaccttgt cgagacaaga agcattgttg tcaaaatccc ggcgcgaggg cgaagtatat tttgaagctg taatcatgtt gttaccgtgt agaacat tgg ccaacccggg ttaataaggc atcacaatac agacgatatc ctcgtttcaa tatgaagagg ggtagagttt tcaaaaagtc cttttaaatg gctgccagac ttcgagcacg gtaccgtcaa actgtatttc ctccagcacc atttttgtgt t tggagagt t cccgtaagac aactgcaccc gatgatttcc tgacactgat catcagaacc ac tctatgtg agctgagtct gc ttgggcaa caacgattac cactatggca tatcactgca gaaagttctg ttcctcaatc gacatgctca tgactaaaac tccgtatacc ctttaaaact attaataaga tatgatacaa agttgcaacc aatagaaatt gaagcttcat agcattcaag atcatcatcc tcaaggagcg ataagaagag taaagacata gacagtagtt taactgatag ttgtggatca aaggactcgc agcaagagga caagacggca atagaggatt ctgtgatgac tttatgtgag gatcaggcag acagtgataa acaatgggac ttcttgaata ctgtcgttgt agataatgag tggtctgatg actatataac tatcatcata tattcagtac tgtgcatgat ccc taacaga 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000 1040 1080 1120 1160 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 8 tcgat 1165 <210> 8 <211> <212> DNA <213> artificial sequence <220> <223> /note= "forward primer for VP24" <400> 8 gggatcgatc tccagacacc aagcaagacc <210> 9 <211> 33 <212> DNA <213> artificial sequence <220> <223> /note= "reverse primer for VP24" <400> 9 gggatcgatg agtcagcata tatgagttag ctc 33 <210> <211> <212> DNA <213> artificial sequence <220> <223> /note= "forward primer for <400> cccatcgatc agatctgcga accggtagag SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 9 <210> 11 <211> 31 <212> DNA <213> artificial sequence <220> <223> /note= "reverse primer for <400> 11 cccatcgatg taccctcatc agaccatgag c 31 <210> 12 <211> 33 <212> DNA <213> artificial sequence <220> <223> /note= "forward primer for <400> 12 gggatcgata gaaaagctgg tctaacaaga tga 33 <210> 13 <211> 36 <212> DNA <213> artificial sequence <220> <223> /note= "reverse primer for <400> 13 cccatcgatc tcacaagtgt atcattaatg taacgt 36 <210> 14 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 <211> <212> DNA <213> artificial sequence <220> <223> /note= "forward primer for <400> 14 cccatcgatc ctacctcggc tgagagagtg <210> <211> 33 <212> DNA <213> artificial sequence <220> <223> /note= "reverse primer for <400> cccatcgata tgttatgcac tatccctgag aag 33 <210> 16 <211> <212> DNA <213> artificial sequence <220> <223> /note= "reverse primer for VP30#2" <400> 16 cccatcgatc tgttagggtt gtatcatacc <210> 17 <211> 676 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCTIUS99/1 4311 <212> PRT <213> Ebola Zaire <220> <400> 17 Met Gly 1 Arg Asp 2 Leu Trp Phe Ser I Ser Thr I Leu Val C Asn Gin L Glu Gly A Ser Ala T Gly Val P Ala Gly G Leu Glu I Cys Leu P: Gly Phe P: Val Ser G Phe Ala P1 Leu Tyr Az Tyr Arg GI Val Ala PI Lys Asp P1.

Glu Pro Va Ser Gly 'ry la rc lu le :*o Ly et .sr: hx ro lu le ro ro ly ie ee ee rr 1 Thr Gly Ile Leu Gin Leu Pro 5 g Phe 1 Ile e Pro .i Gin Arg 1 Arg Gly Lys Pro Trp Lys Ala Arg Thr His Arg Thr Leu Phe Asn 2 Tyr Ly 1! le 2! Let 3! Val 4! Asr Sex Val Arg Lys 95 Ala 105 Lys 115 Ala 125 Cys 135 Gly 145 Lys 155 Leu 165 rhr 175 Ile 3er la er s Arg 5 e Leu Gly Ser Lys Val Ala Trp Val Glu Pro Pro Arg Pro Glu Ala Phe 2 Leu I Ser I Thr C Thr r Thl PhE Val Asr Leu Gly Thr Gly Val Asn Asp Asp Tyr Cys 31y 'er kla ?ro is lu .'hr Sen Gl lE Val Ser Leu Asp Phe Asn Cys Gly Gly Val Ala Ala Thr Glu Gln Pro Asp Ile r Ph~ i Arc HiE Asr Sex Asn Val Arg Tyr Tyr Ser Ile His Gly Phe Val Gly Ala Leu Pro Arg R Phe 1 Thr Asn Lys Thr Leu Pro Ser Glu 100 Asn 110 Glu 120 Arg 130 Lys 140 Asp 150 Phe 160 Ile 170 Val 180 Lys 190 Arg 200 Ser 210 Tyr 220 Gin Ala Thr Gly Phe Giy Thr Asn Giu Thr 225 230 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCTIVS99/1 4311 Glu Tyr Leu Phe Glu Val As Tyr Gin Tyr Gly Ile Trp Ile Val Gin Gly Ile Val Ala Ile Lys Thr Ala Thr I Pro S Lys P Thr P Ser P Asn A Glu G Leu I Val Gin Le Ph Th Ly Asi Arc Sex Sex Thr Met Gin Val Ser Pro ?ro [hr ~sp jer la sp 'ro sn lu le Leu Ser Leu Thr 1 Thr Ser Asn Pro Asn Ala Vai Ser Thr Gly Vai Gin Asn 2 Ala 'J Glu I Phe I Gin;P Asn I Ser A Thr A Le Gi' Il Th Lye Gl.

Gij Ala Thr Ser His His Ser Pro Iyr lal ksp P'hr ~sn Jeu sn 'hr la sn 235 u Glu 245 u Gin 255 y Lys 265 eTrp 275 c Ile 285 5 Lys 295 Glu 305 Ala 315 Arg 325 Thr 335 Glu 345 Ser 355 Leu 365 Pro 375 Asp 385 Lys i 395 Glu 405 Ser 415 Thr 1 425 Thr 1 435 Asp 1 445 His S 455 His H 465 Ser S 475 Thr I 485 Gly G 495 Ser Leu Arg Lys Gly Asn Leu Lys Thr Thr Asn Gin Thr ln ksn Leu I 31n I hr P2 la P ~sn T 'ro A ;er G [is G er G le A Ar As Se Va Gl Le Se Asr Sez GlL Ser 3ly 'hr 3er 3er ~sp 'is la la 'hr la lu in ly la p Asn g Phe n Glu r Asn 1 Asn a Trp i Thr Phe 1 Ile Ser Asp Ser Arg Leu 2 Leu Thr I Ile S His T Ser P Gly P Ser L Thr T Thr A Asp T Lys L Gly V Le Th Th Th Pr Al Ar Th~ Se2 Asr His kla 31u la P'hr iis er Lrg sp 'ro ,ys 'hr la hr eu 1 u Thr 240 r Pro 250 r Ile 260 r Thr 270 o Glu 280 a Phe 290 j Lys 300 :Vai 310 Gly 320 Pro 330 Lys 340 Met 350 Ala 360 .Thr 370 Thr 380 Asn 390 Glu 400 Arg 410 Thr 420 Pro 430 Ser 440 Thr 450 Gly 460 Gly 470 Gly 480 Ala 490 Arg 500 Gly Leu Ile Thr ly Arg Arg Thr SUBSTITUTE SHEET (RULE 26) WO 00/00617 WO 0000617PCT/US99/1 4311 A-rg Giu Ala Ile Val Asn Ala Cys Gir Trp Giy Gin Leu Leu Thr Asp Cys Giu Asp Val Asn Trp Val Ile Asn LAsp Ile Ile Asp Ala Phe Phe Phe His Pro Lys Asp2 Asp Ile Val CysI Prc Gli Prc Tyx Gij Asn Leu Ser Leu Ile His Ile Lys ksn Pro Ile Asr 1Gly Tyr Ile Leu Glu Arg Ile Leu Leu Asp Asp Thr Trp Ala Ala Phe 505 Leu 515 'Ala 525 Phe 535 Giu 545 Ile 555 Thr 565 Ala 575 Leu 585 Gin 595 Gly 605 Trp 615 Gin 625 Leu 635 Trp 645 Gly: 655 Val 665 Val 675 His Ala Gl) Gly Cys Thr Thr Asn Arg Pro Thr Ile Pro rhr Ile Ile ?he Ile Pro Leu Gly Gin Thr Arg Trp Asp Lys Ile Asp Giy Giy Ala Gin Trp Giy Ala Met Leu Ala Giu Lys.

Gly Cys Asn His Gin Trp2 Val 9I Leu I Prc Thr Leu Ala His Arg Leu Leu Ala Gly :ys Ile k.sp rg hr ~he Lys 510 Thr 520 Ala 530 Giu 540 Asn 550 Gin 560 Gin 570 Arg 580 Ile 590 Thr 600 Ile 610 Thr 620 Phe 630 Asp 640 Gin 650 Gly 660 Cys 670 <210> 18 <211> 739 <212> PRT <213> Ebola Zaire <220> <400> 18 Met Asp Ser Arg Pro Gin Lys Ile Trp Met 1 5 Ala Pro Ser Leu Thr Giu Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/S99/1 4311 Val Ile Glu Glu Asp His Leu Gly Arg Leu Lys Glu Leu Leu Lys Glu Trp Phe 2 Lys Met T Val I Phe Leu .Z GlyX Ala I Lys A Gl Pr

IJ-

Ala Se2 Al GlL His Asp Pro Arg Thr Ser Val Val 31n .In krg ?he Jal le jer ~sp ral ys la n Gin o Val Cys n Gly Phe iyr Ser Gly Gly Ala Thr Thr Phe Val Gin Gly Ser Leu I Leu I Ala Ser I Gly I His I Arg I Val I Ala L Gi' TYl G1 Va Let Glr Glj Phe Val Val Leu Glu Ala Gly Arg Leu val 4et jeu ly ~sn ~eu :le ~eu ,ys leu y Ile Val Ar r Gin .i Leu 1 Asp i Leu 1 Gly Ala Arg 105 Lys 115 Ser 125 Ala 135 Ala 145 Ser 155 Glu 165 Gin 175 Ile 185 Gly 195 Arg 205 Ile i 215 His 225 Ser 235 Leu 245 Leu 255 His I 265 Asn C 275 Ser 285 Val Ile Phe Met Asp Val Phe Arg Ser Ala Asn Leu Lys Ile Gin His rhr His ksp 7ali lie 1ln ?ro 1u I er I Asl l Gl Let Ty2 LyE Gl.

Le Gl: Met Ala Phe Ala Gin Tyr Met Asn Gin Ala la ial ys eu lal .eu g Gin n Asn e Gin 2 Glu i Cys Lys STyr 1 Val Glu Lys Pro Gly Leu Cys Val Pro Met Phe I Gly Asn I Gin I Lys rj Thr C Ala Asn S Ala L Ar Let Ala Set Let Let Let Lys Glu Asn Glu Gin Pro Leu His rhr Jai .eu 4et ~sp la 7hr ;lu irg ;er lys g Vai 1 Glu 3.Phe Ala i His Phe 1 Glu 100 ;Lys 110 Leu 120 Ile 130 *Glu 140 Phe 150 Lys 160 Glu 170 Ala 180 Ala 190 Ile 200 Ile 210 His 220 Ala 230 Arg 240 Val 250 Arg 260 Thr 270 Phe 280 His 290 Gly Giu Tyr Aia Pro Phe Aia Arg Leu Leu SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/1 4311 Asn Leu Ser Gly Gly Leu Leu Gin Glu Arg Gin Gin Thr.

Arg Ala Tyr GlyJ His C Asp Pro I Ser '1 Asp I Asp A Arg S Ser G GinT Pro G Ser A Asn G Met L Le Gi' Al Gl Ly Gl Gl Lys Asr Leu Ala ksp Pro ,ln Phr ~sp 'yr sp sp er in ,hr ly la lu eu u Phe y Val a Giy 1i Leu 5 Gin 1 Leu a Lys Lys Ala Ala Ser Asp.

Ile Asp Thr Asp Ser Leu Glu I Thr I Lys c Gin S Pro F Pro L Pro S Thr P Pro Ala Val Arg Leu Asp Lys Asn Met Lys Leu Asp.

Asn.

Asp Tie ly 3iu 2 al I sp 'J ,ys C iy C [is A ,eu 'I er G ro I 29 Va Gla 31! Th~ 32! Asr 33! Glt 345 Glr.

355 His 365 Ile 375 Glu 385 Val 395 Leu 405 Pro 415 Asp 425 Asp 435 ksp 445 Pro 455 Ser 465 ksn 175 .eu 185 'hr 195 ly in i15 Lrg Lrg '35 'hr .45 ly 55 le 5 1 Asn Asn Leu 5 a Leu Ser Ala 5 f Ala His Gly aVai Giy Glu a Ala Ala Thr Gin Tyr Ala Leu Giy Leu Leu Met Asn Ile Ser Phe Thr Leu Arg Thr Glu Ala Lys Thr Ser Asp Ile Pro Asp Asp Asn i Pro Thr Asp Asp Vai Vai l Tyr Gly Giu 'J Gly Met Asn I Phe Asp Leu I Lys Pro Val I Gly Gin Gin I His Ile Giu G Pro Ile Gin A Thr Ile His H Asp Asn Asp A Ser Thr Ser P Asn Giu Giu A Gl 11 Ilt Se~ Gli Ghi Gl.

Asr Phe Gin Lys Ile ly Phe Pro 3er Jal Cyr la ~sp 'ro Jys ;ly sn .is rg ro la 300 u His 310 e Ala 320 r Thr 330 i Tyr 340 a Ala 350 a Ser 360 Asp 370 His 380 Gin 390 Glu 400 Thr 410 His 420 Pro 430 Gly 440 Gin 450 Asp 460 Gin 470 Pro 480 Glu 490 Asn 500 Asn 510 Arg 520 Val 530 Ala 540 Arg 550 Arg 560 Asp SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 16 565 570 Pro Leu Asp Asp Ala Asp Asp Glu Thr Ser 575 580 Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu 585 590 Glu Gin Asp Arg Asp Gly Thr Ser Asn Arg 595 600 Thr Pro Thr Val Ala Pro Pro Ala Pro Val 605 610 Tyr Arg Asp His Ser Glu Lys Lys Glu Leu 615 620 Pro Gin Asp Glu Gin Gin Asp Gin Asp His 625 630 Thr Gin Glu Ala Arg Asn Gin Asp Ser Asp 635 640 Asn Thr Gin Ser Glu His Ser Phe Glu Glu 645 650 Met Tyr Arg His Ile Leu Arg Ser Gin Gly 655 660 Pro Phe Asp Ala Val Leu Tyr Tyr His Met 665 670 Met Lys Asp Glu Pro Val Val Phe Ser Thr 675 680 Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp 685 690 Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu 695 700 Thr Glu Lys Glu Ala Met Asn Glu Glu Asn 705 710 Arg Phe Val Thr Leu Asp Gly Gin Gin Phe 715 720 Tyr Trp Pro Val Met Asn His Lys Asn Lys 725 730 Phe Met Ala Ile Leu Gin His His Gin 735 <210> 19 <211> 251 <212> PRT <213> Ebola Zaire <220> <400> 19 Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu 1 5 Ile Ser Pro Lys Lys Asp Leu Glu Lys Gly Val Val Leu Ser Asp Leu Cys Asn Phe Leu SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCTIUS99/1 4311 Val Ser Gin Thr Ile Gin Gljp Tyr His Lys Met Gin Leu Gly Leu Leu Thr Val Ser Ile Asn Ile Thr.

Leu Arg Leu Thr Trl Lys Thi Thr Asn Trp Ile Ile Ile Asn Lys Leu Asn Tyr Gly Arg Gln Met Leu 31n Ala Gly Asn *Arg Pro Ala Gin Glu Ser His Glu Ile Lys Asn Thr Thr Glu Lys His Gly Gi) Met Asp Asn Asn Leu Asp Pro Asp Phe Gin Arg Leu Gly Gin Asn Pro Pro Glu Ser Ile Ala Phe Leu Ser Arg Gin 105 Leu 115 Trp 125 Asn 135 Leu 145 Ser 155 Asp.

165 Leu 175 Asn 185 Met 195 Asp 205 Gly 215 Ser 225 Ser 235 Glu Leu Ala Phe Thr Val Leu Ala Leu Met Ser Asn Ala Leu His Gly Lys Pro rhr 1hr Phe Leu Pro Pro Ile Ile Ile Gly Leu Arg Leu Ile Leu Ser Thr Phe i Ser Ala Leu I Arg I TrI Asi Hic Ale His Glu Leu Asp Ala Thr Thr Lys Leu His Ser lie leu kla -ys ys let Lys Val 3 Arg Trp Leu Ser Ala Gin Leu Thr Gin Met Lys Val Ile Ile Vai Met 2 Phe Ala I Gin I Val Thr Leu Ser Phe Pro Ala 100 Ser 110 Gly 120 Asn 130 Arg 140 Leu 150 Phe 160 Val 170 Glu 180 Ile 190 .lu 200 %sn 3er ?he 230 ;er 240 Leu lie Leu Giu Phe Asn Ser Ser Leu Ala Ile 245 250 <210> <211> 324 <212> PRT SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/1 4311 18.

<213> Ebola Zaire <220> <400> Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro 1 5 Arg Ala Ala Arg Gin His Ser Arg Asp Gly His Asp His His Val Arg Ala Arg Ser Ser Ser Arg Glu Asn Tyr Arg Gly Glu Tyr Arg Gin Ser Arg Ser Ala Ser Gin Val Arg Val Pro Thr Val Phe His Lys Lys Arg Val Glu Pro Leu Thr Val Pro Pro Ala Pro Lys Asp Ile Cys Pro Thr Leu Lys Lys Gly Phe Leu Cys Asp Ser Ser Phe Cys Lys Lys Asp His Gin Leu Glu Ser Leu Thr Asp Arg Glu Leu 100 Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly 105 110 Ser Val Glu Gin Gin Leu Asn Ile Thr Ala 115 120 Pro Lys Asp Ser Arg Leu Ala Asn Pro Thr 125 130 Ala Asp Asp Phe Gln Gin Glu Glu Gly Pro 135 140 Lys Ile Thr Leu Leu Thr Leu Ile Lys Thr 145 150 Ala Glu His Trp Ala Arg Gin Asp Ile Arg 155 160 Thr Ile Glu Asp Ser Lys Leu Arg Ala Leu 165 170 Leu Thr Leu Cys Ala Val Met Thr Arg Lys 175 180 Phe Ser Lys Ser Gin Leu Ser Leu Leu Cys 185 190 Glu Thr His Leu Arg Arg Glu Gly Leu Gly 195 200 Gin Asp Gin Ala Glu Pro Val Leu Glu Val 205 210 Tyr Gin Arg Leu His Ser Asp Lys Gly Gly 215 220 Ser Phe Glu Ala Ala Leu Trp Gin Gin Trp 225 230 Asp Leu Gin Ser Leu Ile Met Phe Ile Thr 235 240 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 19 Ala Phe Leu Asn Ile Ala Leu Gin Leu Pro 245 250 Cys Glu Ser Ser Ala Val Val Val Ser Gly 255 260 Leu Arg Thr Leu Val Pro Gin Ser Asp Asn 265 270 Glu Glu Ala Ser Thr Asn Pro Gly Thr Cys 275 280 Ser Trp Ser Asp Glu Gly Thr Ser Ile Gin 285 290 Gin Gin Leu Ala Ser Cys Leu His Arg Thr 295 300 Arg Gly Asp Trp His Ala Ala Leu Lys Phe 305 310 Leu Phe Tyr Phe Ser Phe Leu Phe Arg Ile 315 320 Gly Phe Cys Phe <210> 21 <211> 340 <212> PRT <213> Ebola Zaire <220> <400> 21 Met Thr Thr Arg Thr Lys Gly Arg Gly His 1 5 Thr Ala Ala Thr Thr Gin Asn Asp Arg Met Pro Gly Pro Glu Leu Ser Gly Trp Ile Ser Glu Gin Leu Met Thr Gly Arg Ile Pro Val Ser Asp Ile Phe Cys Asp Ile Glu Asn Asn Pro Gly Leu Cys Tyr Ala Ser Gin Met Gin Gin Thr Lys Pro Asn Pro Lys Thr Arg Asn Ser Gin Thr Gin Thr Asp Pro Ile Cys Asn His Ser Phe Glu Glu Val Val Gin Thr Leu Ala Ser Leu Ala Thr Val Val Gin Gin Gin 100 Thr Ile Ala Ser Glu Ser Leu Glu Gin Arg 105 110 Ile Thr Ser Leu Glu Asn Gly Leu Lys Pro SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 115 Val Tyr Asp Met Ala Lys Th2 r Leu Lys Arg Ala Pro Ile Thr Asn Glu Ala His Gin Lys Ala Asp ThrI Ala I Gly2 Leu I Asp I Gin 0 Asi Tyz Ala Tyr Gly Arg Val Asn Glu Lys Leu Leu Asp G1u Ser ys ?ro ksp ~rg ~rg isp i Arg Asp Thr Trp Pro *Gly Pro Leu Asn Asp Pro Val Ser Phe 4 Pro Arg Pro Ile I Pro Gly 9 Gly I Val Leu Ala Ala Ser Lys Gin Asn Phe Leu Gly Gin Asn in.

Val Jai ?ro Jal i rrp YS 'J 12! Cy~ 13! Let 14! Th 151 Gli 161 Lei 175 Ile 185 Sex 195 Ser 205 Gly 215 Arg 225 Phe 235 Val 245 Ser 255 Ala 265 Cys 275 Pro 285 Ile 295 krg 305 ?ro 315 [al 325 rhr 135 5 s Al i Va .Ale 1 His STyx Glu Val Thr Lys Asn Gly Ile Leu Ser Ala Ile His Ala Pro Cys Leu Glu i Met Ala Gly Glu Ser Arg Thr Pro Ile Thr Cys Asp Leu Leu Phe Ile I Cys C Ser I Val I Gly L Il Mel Th Al Glr Gil Arg Glu Ser Asp Met Ala Lys lie kla Ile .In ~rg In 'ro 'he zeu e Ser t Val r Thr i Thr 1 Pro 1 Ser rAsp Ala Leu Ile Tyr Phe Leu 4 Ile Glu Gin Asp I Ser I Lys c Lys I 3 Ln L 3 Lys I 3 120 Ser 130 Ala 140 Gly 150 Glu 160 Pro 170 Ala 180 Glu 190 Phe 200 Thr 210 Ser 220 Asp 230 His 240

G

1 y 250 His 260 ;iy 270 Ile 180 kla ~rg 100 er 110 :le ~eu le <210> 22 <211> 326 <212> PRT <213> Ebola Zaire SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 <220> <400> Met Arc 1 Pro Glu Arg Ser Asn Ser Ser Val Leu Arg His Ala Ser Ala Val Ile Gin Ile Ala Asp Thr Thr Thr Ile Pro Leu Gly Ile Arg Ile Phe Val Tyr Phe Leu Ile Trp Thr Gly Ala Pro Lys Lys Ser Leu Thr 22 g Arg Val Ile Leu Pro 5 STyr Met Glu Ala Ile Asn Ser Thr Ile Ala Asn Thr Gly Phe Leu Asn Gly Asp Thr Pro Pro Ile Ala Asp Asp Ser His Thr Pro Gly Phe Ile Leu Glu Ala Ser Gly Pro Lys Val Pro Ile Trp Leu Pro Gin Lys Thr Tyr Ser 105 Ala Ala Ile Met Leu 115 Thr His Phe Gly Lys 125 Val Arg Val Asn Arg 135 Pro Asp His Pro Leu 145 Gly Asn Gin Ala Phe 155 Leu Pro Pro Val Gin 165 Thr Phe Asp Leu Thr 175 Thr Gin Pro Leu Pro 185 Asp Asp Thr Pro Thr 195 Leu Arg Pro Gly Ile 205 Leu Arg Pro Ile Leu 215 Gly Lys Lys Gly Asn 225 Ser Pro Glu Lys Ile Thr Ala Pro Tyr Pro Val Arg Gly Gly Thr Pro Glu Ser Thr Ser Met Leu Leu Phe Ala Ala Leu Arg Leu Leu Ala Ala Gly Ser Leu Ser Asn Pro Ile Asp Val Ser SVal Asn SMet Lys Gly Val 100 Asp Ser 110 Ser Tyr 120 Thr Asn 130 Gly Pro 140 Leu Leu 150 Gin Glu 160 Pro Gin 170 Leu Lys 180 Ala Thr 190 Ser Asn 200 Phe His 210 Pro Asn 220 Ala Asp 230 31n Ala Ile 235 Met Thr Ser Leu Gin 245 Asp Phe Lys 240 Ile Val 250 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCTIUS99/14311 22 Pro Ile Asp Pro Thr Lys Asn Ile Met Gly 255 260 Ile Glu Val Pro Glu Thr Leu Val His Lys 265 270 Leu Thr Gly Lys Lys Val Thr Ser Lys Asn 275 280 Gly Gin Pro Ile Ile Pro Val Leu Leu Pro 285 290 Lys Tyr Ile Gly Leu Asp Pro Val Ala Pro 295 300 Gly Asp Leu Thr Met Val Ile Thr Gin Asp 305 310 Cys Asp Thr Cys His Ser Pro Ala Ser Leu 315 320 Pro Ala Val Ile Glu Lys 325 <210> 23 <211> 288 <212> PRT <213> Ebola Zaire <220> <400> 23 Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro 1 5 Arg Ala Ala Arg Gin His Ser Arg Asp Gly His Asp His His Val Arg Ala Arg Ser Ser Ser Arg Glu Asn Tyr Arg Gly Glu Tyr Arg Gin Ser Arg Ser Ala Ser Gin Val Arg Val Pro Thr Val Phe His Lys Lys Arg Val Glu Pro Leu Thr Val Pro Pro Ala Pro Lys Asp Ile Cys Pro Thr Leu Lys Lys Gly Phe Leu Cys Asp Ser Ser Phe Cys Lys Lys Asp His Gin Leu Glu Ser Leu Thr Asp Arg Glu Leu 100 Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly 105 110 Ser Val Glu Gin Gin Leu Asn Ile Thr Ala 115 120 Pro Lys Asp Ser Arg Leu Ala Asn Pro Thr 125 130 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US99/14311 23 Ala Asp Asp Phe Gin Gin Glu Glu Gly Pro 135 140 Lys Ala Thr Leu Phe Glu Gin Tyr Ser Asp Ala Cys Leu Glu Ser Ile Thr Leu Leu 145 Glu His Trp Ala 155 Ile Glu Asp Ser 165 Thr Leu Cys Ala 175 Ser Lys Ser Gin 185 Thr His Leu Arg 195 Asp Gin Ala Glu 205 Gin Arg Leu His 215 Phe Glu Ala Ala 225 Arg Gin Ser Leu 235 Phe Leu Asn Ile 245 Glu Ser Ser Ala 255 Arg Thr Leu Val 265 Glu Ala Ser Thr 275 Trp Ser Asp Glu 285 Thr Leu Ile Arg Gin Asp Lys Leu Arg Val Met Thr Leu Ser Leu Arg Glu Gly Pro Val Leu Ser Asp Lys Leu Trp Gin Ile Met Phe Ala Leu Gin Val Val Val Pro Gin Ser Asn Pro Gly Gly Thr Pro Lys Thr 150 Ile Arg 160 Ala Leu 170 Arg Lys 180 Leu Cys 190 Leu Gly 200 Glu Val 210 Gly Gly 220 Gin Trp 230 Ile Thr 240 Leu Pro 250 Ser Gly 260 Asp Asn 270 Thr Cys 280 <210> 24 <211> 11 <212> PRT <213> Ebola Zaire <220> <400> 24 Val Tyr Gin Val Asn Asn Leu Glu Glu Ile 1 5 Cys <210> <211> 23 SUBSTITUTE SHEET (RULE 26) WO 00/00617 PCT/US991431 I 24.

<212> PRT <213> Ebola Zaire <220> <400> Leu Lys Phe Ie Asn Lys Leu Asp Ala Leu 1 5 Leu Val Val Asn Tyr Asn Gly Leu Leu Ser Ser Ie Phe SUBSTITUTE SHEET (RULE 26)

Claims (37)

1. A DNA fragment which encodes a VP24 Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO: 3, or a polynucleotide fragment comprising at least 15 nucleotides and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

2. A DNA fragment which encodes a VP30 Ebola protein, said DNA fragment comprising the sequence specified in any of SEQ ID NO: 4 and SEQ ID NO: 7, or a polynucleotide fragment comprising at least 15 nucleotides and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

3. A DNA fragment which encodes a VP35 Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO: 5, or a polynucleotide fragment comprising at least 15 nucleotides and which upon expression in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal. 20 4. A DNA fragment which encodes a VP40 Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO: 6, or a polynucleotide fragment comprising at least 15 nucleotides and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

5. A DNA fragment which encodes a VP24 Ebola protein, said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO: 19, or a conservative substitution thereof and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

6. A DNA fragment which encodes a VP30 Ebola protein, said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in any of SEQ ID NO: 20 and SEQ ID NO: 23, or a conservative substitution thereof and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

7. A DNA fragment which encodes a VP35 Ebola protein, said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO: 21, or a conservative substitution thereof and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

8. A DNA fragment which encodes a VP40 Ebola protein, said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO: 22, or a conservative substitution thereof and which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal. *o*o

9. The DNA fragment of any one of claims 1 to 8 wherein said animal is a o mammal. A recombinant DNA construct comprising: a VEE viral vector, and (ii) at least one of the Ebola virus DNA fragments chosen from the group consisting of SEQ ID NO: 3, 4, 5, 6 and 7 or a fragment thereof comprising at least "-.nucleotides which upon expression in an animal produces a protective cytotoxic T S"lymphocyte response or antibody response to Ebola virus in said animal.

11. A recombinant DNA construct comprising: a VEE viral vector, and (ii) at least one of the Ebola virus DNA fragments encoding a peptide chosen from the group consisting of SEQ ID NO: 19, 20, 21, 22, 23, 24 and 25 or a conservative substitution thereof which upon expression in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

12. The recombinant DNA construct of any one of claims 10 and 11 wherein said animal is a mammal.

13. The recombinant DNA construct according to claim 10 wherein said Ebola virus DNA fragments are from Ebola Zaire 1976.

14. The recombinant DNA construct according to claim 13 wherein said construct is VRepEboVP24. The recombinant DNA construct according to claim 13 wherein said construct is

16. The recombinant DNA construct according to claim 13 wherein said construct is

17. The recombinant DNA construct according to claim 13 wherein said construct is

18. The recombinant DNA construct according to claim 13 wherein said construct is for VRepEboVP30

19. Self replicating RNA produced from a construct chosen from the group consisting S ofEboVP24ReP, EboVP30ReP, EboVP35ReP, EboVP40ReP and EboVP30ReP which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal. 48 The self-replicating RNA of claim 19 wherein said animal is a mammal.

21. Infectious alphavirus particles produced from packaging the self replicating RNA of claim 19.

22. A pharmaceutical composition comprising infectious alphavirus particles according to claim 21 in an effective immunogenic amount in a pharmaceutically acceptable carrier and/or adjuvant.

23. A host cell transformed with a recombinant DNA construct according to claim

24. A host cell transformed with a recombinant DNA construct according to claim 11. A host cell according to claim 23 wherein said host cell is prokaryotic.

26. A host cell according to claim 23 wherein said host cell is eukaryotic.

27. A host cell according to claim 24 wherein said host cell is prokaryotic.

28. A host cell according to claim 24 wherein said host cell is eukaryotic.

29. A method for producing Ebola virus proteins comprising culturing the cells according to claim 23 under conditions such that said DNA fragment is expressed and said Ebola protein is produced. A method for producing Ebola virus proteins comprising culturing the cells according to claim 24 under conditions such that said DNA fragment is expressed and *o said Ebola protein is produced.

31. A method for producing Ebola virus proteins comprising culturing the cells according to claim 25 under conditions such that said DNA fragment is expressed and said Ebola protein is produced.

32. A method for producing Ebola virus proteins comprising culturing the cells according to claim 26 under conditions such that said DNA fragment is expressed and said Ebola protein is produced.

33. An isolated and purified Ebola VP24 protein specified in SEQ ID NO: 19 and conservative substitutions thereof, or an immunologically identifiable portion thereof which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

34. An isolated and purified Ebola VP30 protein specified in any of SEQ ID NO: and SEQ ID NO: 23 and conservative substitutions thereof, or an immunologically identifiable portion thereof which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

35. An isolated and purified Ebola VP35 protein specified in SEQ ID NO: 21 and conservative substitutions thereof, or an immunologically identifiable portion thereof which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal. 20 36. An isolated and purified Ebola VP40 protein specified in a SEQ ID NO: 22 and conservative substitutions thereof, or an immunologically identifiable portion thereof which upon expression in a VEE viral vector in an animal produces a protective cytotoxic T lymphocyte response or antibody response to Ebola virus in said animal.

37. The isolated protein of any one of claims 33 to 36 wherein said animal is a 0 *mammal.

38. The isolated protein of any one of claims 33 to 36 wherein said mammal is a human.

39. An antibody to a peptide encoded by the sequence specified in SEQ ID NO: 19, 21, 22, 23, 24 and A method for detecting Ebola virus infection comprising contacting a sample from a subject suspected of having Ebola virus infection with a antibody according to claim 39 and detecting the presence or absence by detecting the presence or absence of a complex formed between the Ebola protein and antibodies specific therefor.

41. A vaccine for Ebola comprising alphavirus particles of claim 21.

42. A method for the diagnosis of Ebola virus infection comprising the steps of: contacting a sample from an individual suspected of having Ebola virus infection with an antibody to Ebola proteins according to claim 39; and (ii) detecting the presence or absence of Ebola virus infection by detecting the presence or absence of a complex formed between Ebola proteins and antibodies specific therefor.

43. A pharmaceutical composition comprising the self replicating RNA of claim 19 I in an effective immunogenic amount in a pharmaceutically acceptable carrier and/or adjuvant.

44. A pharmaceutical composition comprising one or more recombinant DNA constructs chosen from the group consisting of VRepEboVP24, VRepEboVP40 and VRepEboVP30 in a pharmaceutically acceptable amount, in a pharmaceutically acceptable carrier and/or adjuvant, which 25 upon administration to an animal produces a protective cytotoxic T lymphocyte Sresponse or antibody response to Ebola virus in said animal. *o 0 45. The pharmaceutical composition of claim 40 wherein said animal is a mammal.

46. A pharmaceutical composition comprising a peptide encoded by any of SEQ ID 46. A pharmaceutical composition comprising a peptide encoded by any of SEQ ID NO:24 and SEQ ID NO:25 and expressed in a VEE viral vector, in a pharmaceutically acceptable amount, in a pharmaceutically acceptable carrier and/or adjuvant. 0 0* *004. 04